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Sample records for 17-allylamino-17-demethoxygeldanamycin 17-aag tanespimycin

  1. Acquired resistance to 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) in glioblastoma cells

    PubMed Central

    Gaspar, Nathalie; Sharp, Swee Y; Pacey, Simon; Jones, Chris; Walton, Michael; Vassal, Gilles; Eccles, Suzanne; Pearson, Andrew; Workman, Paul

    2009-01-01

    HSP90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) which is currently in phase II/III clinical trials, are promising new anticancer agents. Here, we explored acquired resistance to HSP90 inhibitors in glioblastoma, a primary brain tumor with poor prognosis. Glioblastoma cells were exposed continuously to increased 17-AAG concentrations. Four 17-AAG-resistant glioblastoma cell lines were generated. High resistance levels with resistance indices (RI=resistant line IC50/parental line IC50) of 20-137 were obtained rapidly (2-8 weeks). After cessation of 17-AAG exposure, RI decreased and then stabilised. Cross-resistance was found with other ansamycin benzoquinones but not with the structurally unrelated HSP90 inhibitors, radicicol, the purine BIIB021 and the resorcinylic pyrazole/isoxazole amide compounds VER-49009, VER-50589, and NVP-AUY922. An inverse correlation between NQO1 expression/activity and 17-AAG IC50 was observed in the resistant lines. The NQO1 inhibitor ES936 abrogated the differential effects of 17-AAG sensitivity between the parental and resistant lines. NQO1 mRNA levels and NQO1 DNA polymorphism analysis indicated different underlying mechanisms: reduced expression and selection of the inactive NQO1*2 polymorphism. Decreased NQO1 expression was also observed in a melanoma line with acquired resistance to 17-AAG. No resistance was generated with VER-50589 and NVP-AUY922. In conclusion, low NQO1 activity is a likely mechanism of acquired resistance to 17-AAG in glioblastoma, melanoma and possibly other tumor types. Such resistance can be overcome with novel HSP90 inhibitors. PMID:19244114

  2. Identification of genes involved in the sensitivity to antitumour drug 17-allylamino,17-demethoxygeldanamycin (17AAG).

    PubMed

    Barresi, Vincenza; Fortuna, Cosimo G; Garozzo, Roberta; Musumarra, Giuseppe; Scirè, Salvatore; Condorelli, Daniele F

    2006-05-01

    In the present study we analysed the gene expression database provided by the National Cancer Institute in an attempt to correlate activity profiles of geldanamycin, 17AAG and 11 other analogues in 60 human tumor cell lines with their gene expression profiles determined by the cDNA microarray technique. On the basis of the activity profiles two classes of geldanamycin analogues could be distinguished, having geldanamycin and 17AAG, respectively, as prototype compounds (denominated as gelda-like and 17AAG-like classes). Application of the "soft" statistical methodology of PLS (partial least squares modelling in latent variables or projections to latent structures) allowed us to evaluate the influence of each gene expression target in determining the therapeutical responses. The transcript encoding the translocating chain-associated membrane protein (TRAM) showed a significant statistical correlation with activity profiles of 17AAG. In order to validate the role of TRAM in determining sensitivity to 17AAG we induced a selective knocking-down of this transcript by the RNA interference methodology in H226 non-small cell lung carcinoma cell line. The efficiency of double-stranded RNA oligonucleotides (short-interfering RNAs, siRNAs) was determined by measuring TRAM mRNA levels by quantitative real-time RT-PCR at different times (24-72 hours) after siRNA lipotransfection. A significant increase in chemosensitivity to 17AAG was observed in siRNA-silenced cells. Although a number of factors may affect tumour sensitivity to 17AAG the present methodology allowed us to dissect out a single parameter which may be partly responsible for its activity. PMID:16880941

  3. The effect of 17-allylamino-17-demethoxygeldanamycin alone or in combination with paclitaxel on anaplastic thyroid carcinoma cells.

    PubMed

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2015-04-01

    The effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an hsp90 inhibitor, alone or in combination with paclitaxel on survival of anaplastic thyroid carcinoma (ATC) was evaluated. In 8505C and CAL62 cells, after treatment of 17-AAG, cell viability decreased, and the percentage of dead cells increased. 17-AAG did not cause cleavage of caspase-3 protein, and change expression of IAPs. Pretreatment of z-VAD-fmk did not alter cell viability and the percentage of dead cells. In 17-AAG-treated cells, knockdown of p53 rescued growth inhibition, while cycloheximide attenuated cell death. When cells were treated with both 17-AAG and paclitaxel, all of the combination index values were higher than 1, indicating antagonism between 17-AAG and paclitaxel. In 17-AAG- and paclitaxel-treated cells, compared with paclitaxel alone-treated cells, the protein levels of hsp90, hsp70, and hsc70 increased. In conclusion, our results suggest that 17-AAG induces non-apoptotic cell death requiring de novo protein synthesis in ATC cells. Moreover, these results demonstrate that 17-AAG antagonizes paclitaxel with concomitant alterations in hsp90 client proteins in ATC cells. PMID:25096912

  4. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

    SciTech Connect

    Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi . E-mail: rokayasu@nirs.go.jp

    2006-12-22

    In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.

  5. Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90.

    PubMed

    Sydor, Jens R; Normant, Emmanuel; Pien, Christine S; Porter, James R; Ge, Jie; Grenier, Louis; Pak, Roger H; Ali, Janid A; Dembski, Marlene S; Hudak, Jebecka; Patterson, Jon; Penders, Courtney; Pink, Melissa; Read, Margaret A; Sang, Jim; Woodward, Caroline; Zhang, Yilong; Grayzel, David S; Wright, Jim; Barrett, John A; Palombella, Vito J; Adams, Julian; Tong, Jeffrey K

    2006-11-14

    Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90. PMID:17090671

  6. 17-Allylamino-17-Demethoxygeldanamycin and the Enhancement of PS-341-Induced Lung Cancer Cell Death by Blocking the NF-κB and PI3K/Akt Pathways.

    PubMed

    Lee, Kyoung-Hee; Jang, An-Hee; Yoo, Chul-Gyu

    2015-09-01

    PS-341 is a highly selective and potent proteasome inhibitor that is cytotoxic to various types of cancer. However, no objective response was seen in a clinical trial with PS-341 as a single agent in non-small cell lung cancer. Its antitumor activity is limited by the simultaneously activated antiapoptosis pathway. Recently, PS-341-induced NF-κB activation via IκBα degradation has been suggested to be one of its antiapoptotic effects. In this study, we investigated the effects of a combined application of the heat shock protein (Hsp) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) with PS-341 in lung cancer cells. Hsp90 inhibition with 17-AAG was effective in enhancing PS-341-induced lung cancer cell death in vitro and in vivo. 17-AAG pretreatment induced the degradation of upstream regulators of IκB, IL-1R-associated kinase-1 (IRAK-1), and IκB kinases (IKKs), dose and time dependently, which resulted in blocking of PS-341-induced IκBα degradation, p65 nuclear translocation, transcriptional activity, and NF-κB-regulated antiapoptotic gene expressions such as COX-2. The concentrations of 17-AAG used for combinatorial treatment with PS-341 did not change cell viability or the activity of proteasome complex. Moreover, 17-AAG pretreatment decreased the level of phsophorylated Akt at serine 473 residue and suppressed active Akt-dependent inactivation of glycogen synthase kinase 3β. 17-AAG mediated the dissociation of its client proteins (IRAK-1, IKKs, and Akt) from the Hsp90 complex. As a result, it induced degradation of target proteins. Our results suggest that the combination of 17-AAG and PS-341 could be an effective anticancer therapy that overcomes the limited effects of PS-341. PMID:25633180

  7. Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis

    PubMed Central

    Santos, Diego M.; Petersen, Antonio L. O. A.; Celes, Fabiana S.; Borges, Valeria M.; Veras, Patricia S. T.; de Oliveira, Camila I.

    2014-01-01

    Background Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces L. (L.) amazonensis infection in vitro. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with L. (V.) braziliensis. Methodology/Principal findings Exposure of axenic L. (V.) braziliensis promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to L. (V.) braziliensis-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O−2) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated in vivo, employing BALB/c mice infected with L. braziliensis. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability. Conclusion/Significance 17-AAG effectively inhibited the growth of L. braziliensis, both in vitro and in vivo. Given the chronicity of L. (V.) braziliensis infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis. PMID:25340794

  8. Topically applied Hsp90 inhibitor 17AAG inhibits UVR-induced cutaneous squamous cell carcinomas.

    PubMed

    Singh, Anupama; Singh, Ashok; Sand, Jordan M; Bauer, Samuel J; Hafeez, Bilal Bin; Meske, Louise; Verma, Ajit K

    2015-04-01

    We present here that heat-shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits UVR-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500 nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ m(-2)). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and protein kinase C epsilon (PKCɛ)-overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by a decrease in UVR-induced (1) hyperplasia, (2) Hsp90β-PKCɛ interaction, and (3) expression levels of Hsp90β, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473, and matrix metalloproteinase (MMP). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR-exposed or organ transplant population. PMID:25337691

  9. Topically applied Hsp90 inhibitor 17AAG inhibits ultraviolet radiation-induced cutaneous squamous cell carcinomas

    PubMed Central

    Singh, Anupama; Singh, Ashok; Sand, Jordan M.; Bauer, Samuel J.; Hafeez, Bilal Bin; Meske, Louise; Verma, Ajit K.

    2014-01-01

    We present here that Heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ/m2). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and PKCε overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by decrease in UVR-induced: 1) hyperplasia, 2) Hsp90β-PKCε interaction, 3) expression levels of Hsp90β, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473 and matrix metalloproteinase (MMPs). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR exposed or organ transplant population. PMID:25337691

  10. Phase II Trial of Gemcitabine and Tanespimycin (17AAG) in Metastatic Pancreatic Cancer: A Mayo Clinic Phase II Consortium Study

    PubMed Central

    Pedersen, Katrina S.; Kim, George P.; Foster, Nathan R.; Wang-Gillam, Andrea; Erlichman, Charles; McWilliams, Robert R.

    2016-01-01

    Objectives Heat Shock Protein 90 (HSP90) is a molecular chaperone that stabilizes many oncogenic proteins. HSP90 inhibitors may sensitize tumors to cytotoxic agents by causing client protein degradation. Gemcitabine, which has modest activity in pancreas cancer, activates Chk1, a client protein of HSP90. This phase II trial was designed to determine whether 17AAG could enhance the clinical activity of gemcitabine through degradation of Chk1 in patients with stage IV pancreatic cancer. Methods A multicenter, prospective study combining gemcitabine and 17AAG enrolled patients with stage IV pancreatic adenocarcinoma, adequate liver and kidney function, ECOG performance status 0-2, and no prior chemotherapy for metastatic disease. The primary goal was to achieve a 60% overall survival at six months. Sixty-six patients were planned for accrual, with an interim analysis after 25 patients enrolled. Results: After a futility analysis to achieve the endpoint, accrual was halted with 21 patients enrolled. No complete or partial responses were seen. 40% of patients were alive at 6 months. Median overall survival was 5.4 months. Tolerability was moderate, with 65% of patients having ≥ grade 3 adverse events (AE), and 15% having grade 4 events. Conclusions The lack of clinical activity suggests that targeting Chk1 by inhibiting HSP90 is not important in pancreatic cancer sensitivity to gemcitabine alone. Further studies of HSP90 targeted agents with gemcitabine alone are not warranted. PMID:25952464

  11. 17-AAG suppresses growth and invasion of lung adenocarcinoma cells via regulation of the LATS1/YAP pathway

    PubMed Central

    Ye, Xiang-Yun; Luo, Qing-Quan; Xu, Yun-Hua; Tang, Nai-Wang; Niu, Xiao-Min; Li, Zi-Ming; Shen, Sheng-Ping; Lu, Shun; Chen, Zhi-Wei

    2015-01-01

    The large tumour suppressor 1 (LATS1) signalling network has been proved to be an essential regulator within the cell, participating in multiple cellular phenotypes. However, it is unclear concerning the clinical significance of LATS1 and the regulatory mechanisms of 17-Allylamino-17- demethoxygeldanamycin (17-AAG) in lung adenocarcinoma (LAC). The aim of the present study was to investigate the correlation of LATS1 and yes-associated protein (YAP) expression with clinicopathological characteristics in LAC patients, and the effects of 17-AAG on biological behaviours of LAC cells. Subcutaneous LAC tumour models were further established to observe the tumour growth in nude mice. The results showed that the positive expression of LATS1 was significantly lowered (26.7% versus 68.0%, P < 0.001), while that of YAP was elevated (76.0% versus 56.0%, P + 0.03) in LAC tissues compared to the adjacent non-cancerous tissues; LAST1 expression was negatively correlated with YAP expression (r + 0.432, P < 0.001) and lymphatic invasion of the tumour (P + 0.015). In addition, 17-AAG inhibited proliferation and invasion, and induced cell apoptosis and cycle arrest in LAC cells together with increased expression of E-cadherin and p-LATS1, and decreased expression of YAP and connective tissue growth factor. Tumour volumes and weight were much smaller in 17-AAG-treated groups than those in untreated group (P < 0.01). Taken together, our findings indicate that decreased expression of LATS1 is associated with lymphatic invasion of LAC, and 17-AAG suppresses growth and invasion of LAC cells via regulation of the LATS1/YAP pathway in vitro and in vivo, suggesting that we may provide a promising therapeutic strategy for the treatment of human LAC. PMID:25712415

  12. The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells.

    PubMed

    Hirakawa, Hirokazu; Fujisawa, Hiroshi; Masaoka, Aya; Noguchi, Miho; Hirayama, Ryoichi; Takahashi, Momoko; Fujimori, Akira; Okayasu, Ryuichi

    2015-03-01

    Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors. PMID:25582113

  13. Acquired resistance to HSP90 inhibitor 17-AAG and increased metastatic potential are associated with MUC1 expression in colon carcinoma cells.

    PubMed

    Liu, Xin; Ban, Li-Li; Luo, Gang; Li, Zhi-Yao; Li, Yun-Feng; Zhou, Yong-Chun; Wang, Xi-Cai; Jin, Cong-Guo; Ye, Jia-Gui; Ma, Ding-Ding; Xie, Qing; Huang, You-Guang

    2016-06-01

    Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances the survival, growth, and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colon cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. Cross-resistance was found with another HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin. The resistant cells showed obvious morphology changes with a metastatic phenotype and significant increases in migration and adhesion to collagens. Western blotting analysis of epithelial-mesenchymal transition molecular markers found that expression of E-cadherin downregulated, whereas expression of N-cadherin and β-catenin upregulated in the resistant cells. Mucin 1 (MUC1) has been reported to mediate metastasis as well as chemical resistance in many cancers. Here, we found that MUC1 expression was significantly elevated in the acquired drug resistance cells. 17-AAG treatment could decrease MUC1 more in parental cells than in acquired 17-AAG-resistant cells. Further study found that knockdown of MUC1 expression by small interfering RNA could obviously re-sensitize the resistant cells to 17-AAG treatment, and decrease the cell migration and adhesion. These were coupled with a downregulation in N-cadherin and β-catenin. The results indicate that HSP90 inhibitor therapies in colon carcinomas could generate resistance and increase metastatic potential that might mediated by upregulation of MUC1 expression. Findings from this study further our understanding of the potential clinical effects of HSP90-directed therapies in

  14. Assaying Pharmacodynamic Endpoints with Targeted Therapy: Flavopiridol and 17AAG Induced Dephosphorylation of Histone H1.5 in Acute Myeloid Leukemia

    PubMed Central

    Wang, Liwen; Harshman, Sean W.; Liu, Shujun; Ren, Chen; Xu, Hua; Sallans, Larry; Grever, Michael; Byrd, John C.; Marcucci, Guido; Freitas, Michael A.

    2011-01-01

    Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this report we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 Acute Myeloid Leukemia (AML) cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. Liquid chromatography mass spectrometry profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin dependent kinase inhibitor, flavopiridol, and the Hsp90 inhibitor, 17AAG (17-(Allylamino)-17-demethoxygeldanamycin). In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation. PMID:21110323

  15. Design and evaluation of micellar nanocarriers for 17-allyamino-17-demethoxygeldanamycin (17-AAG).

    PubMed

    Chandran, Thripthy; Katragadda, Usha; Teng, Quincy; Tan, Chalet

    2010-06-15

    17-Allyamino-17-demethoxygeldanamycin (17-AAG) is a potent anticancer agent currently undergoing phases I and II clinical trials. However, the clinical development of 17-AAG has been hindered by its poor aqueous solubility and hepatotoxicity. This study aimed to devise novel micellar nanocarriers for 17-AAG that improve its solubility and retain the incorporated drug for a prolonged period of time. We have found that 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]/D-alpha-tocopheryl polyethylene glycol 1000 (PEG-DSPE/TPGS) mixed micelles (at a 1:2 molar ratio) can deliver 17-AAG at clinically relevant doses. By modulating the concentrations of micelle-forming copolymers, the burst release of 17-AAG from PEG-DSPE/TPGS mixed micelles was substantially reduced with a release half-life up to about 8h. Our (1)H NMR spectroscopy results revealed that the incorporation of TPGS into PEG-DSPE micelles restricted internal molecular motions of copolymers in both the corona and core regions of the micelles, leading to the delayed drug release. Cytotoxicity of 17-AAG formulated in PEG-DSPE/TPGS mixed micelles against human ovarian cancer SKOV-3 cells was comparable to that of free 17-AAG. 17-AAG-loaded PEG-DSPE/TPGS mixed micelles may offer a promising alternative to the current 17-AAG formulations for the treatment of solid tumors. PMID:20363305

  16. Design and Evaluation of Micellar Nanocarriers for 17-allyamino-17-demethoxygeldanamycin (17-AAG)

    EPA Science Inventory

    17-Allyamino-17-demethoxygeldanamycin (17-AAG) is a potent anticancer agent currently undergoing phases I and II clinical trials. However, the clinical development of 17-AAG has been hindered by its poor aqueous solubility and hepatotoxicity. This study aimed to devise novel mice...

  17. Hyaluronic acid-decorated poly(lactic-co-glycolic acid) nanoparticles for combined delivery of docetaxel and tanespimycin.

    PubMed

    Pradhan, Roshan; Ramasamy, Thiruganesh; Choi, Ju Yeon; Kim, Jeong Hwan; Poudel, Bijay Kumar; Tak, Jin Wook; Nukolova, Natalia; Choi, Han-Gon; Yong, Chul Soon; Kim, Jong Oh

    2015-06-01

    Multiple-drug combination therapy is becoming more common in the treatment of advanced cancers because this approach can decrease side effects and delay or prevent drug resistance. In the present study, we developed hyaluronic acid (HA)-decorated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (HA-PLGA NPs) for co-delivery of docetaxel (DTX) and tanespimycin (17-AAG). DTX and 17-AAG were simultaneously loaded into HA-PLGA NPs using an oil-in-water emulsification/solvent evaporation method. Several formulations were tested. HA-PLGA NPs loaded with DTX and 17-AAG at a molar ratio of 2:1 produced the smallest particle size (173.3±2.2nm), polydispersity index (0.151±0.026), and zeta potential (-12.4±0.4mV). Approximately 60% and 40% of DTX and 17-AAG, respectively, were released over 168h in vitro. Cytotoxicity assays performed in vitro using MCF-7, MDA-MB-231, and SCC-7 cells showed that dual drug-loaded HA-PLGA NPs at a DTX:17-AAG molar ratio of 2:1 exhibited the highest synergistic effect, with combination index values of 0.051, 0.036, and 0.032, respectively, at the median effective dose. Furthermore, synergistic antitumor activity was demonstrated in vivo in a CD44 and RHAMM (CD168) - overexpressing squamous cell carcinoma (SCC-7) xenograft in nude mice. These findings indicated that nanosystem-based co-delivery of DTX and 17-AAG could provide a promising combined therapeutic strategy for enhanced antitumor therapy. PMID:25843864

  18. Low intensity focused ultrasound (LOFU) modulates unfolded protein response and sensitizes prostate cancer to 17AAG

    PubMed Central

    Saha, Subhrajit; Bhanja, Payel; Partanen, Ari; Zhang, Wei; Liu, Laibin; Tomé, Wolfgang; Guha, Chandan

    2014-01-01

    The hypoxic tumor microenvironment generates oxidative Endoplasmic Reticulum (ER) stress, resulting in protein misfolding and unfolded protein response (UPR). UPR induces several molecular chaperones including heat-shock protein 90 (HSP90), which corrects protein misfolding and improves survival of cancer cells and resistance to tumoricidal therapy although prolonged activation of UPR induces cell death. The HSP90 inhibitor, 17AAG, has shown promise against various solid tumors, including prostate cancer (PC). However, therapeutic doses of 17AAG elicit systemic toxicity. In this manuscript, we describe a new paradigm where the combination therapy of a non-ablative and non-invasive low energy focused ultrasound (LOFU) and a non-toxic, low dose 17AAG causes synthetic lethality and significant tumoricidal effects in mouse and human PC xenografts. LOFU induces ER stress and UPR in tumor cells without inducing cell death. Treatment with a non-toxic dose of 17AAG further increased ER stress in LOFU treated PC and switch UPR from a cytoprotective to an apoptotic response in tumors resulting significant induction of apoptosis and tumor growth retardation. These observations suggest that LOFU-induced ER stress makes the ultrasound-treated tumors more susceptible to chemotherapeutic agents, such as 17AAG. Thus, a novel therapy of LOFU-induced chemosensitization may be designed for locally advanced and recurrent tumors. PMID:25594042

  19. Synergistic Combinations of Multiple Chemotherapeutic Agents in High Capacity Poly(2-oxazoline) Micelles

    PubMed Central

    Han, Yingchao; He, Zhijian; Schulz, Anita; Bronich, Tatiana K.; Jordan, Rainer; Luxenhofer, Robert; Kabanov, Alexander V.

    2012-01-01

    Many effective drugs for cancer treatment are poorly water-soluble. In combination chemotherapy, needed excipients in additive formulations are often toxic and restrict their applications in clinical intervention. Here, we report on amphiphilic poly(2-oxazoline)s (POx) micelles as a promising high capacity delivery platform for multi-drug cancer chemotherapy. A variety of binary and ternary drugs combinations of paclitaxel (PTX), docetaxel (DTX), 17-allylamino-17-demethoxygeldanamycin (17-AAG), etoposide (ETO) and bortezomib (BTZ) were solubilized in defined polymeric micelles achieving unprecedented high total loading capacities of up to 50 wt.% drug per final formulation. Multi-drug loaded POx micelles showed enhanced stability in comparison to single-drug loaded micelles. Drug ratio dependent synergistic cytotoxicity of micellar ETO/17-AAG was observed in MCF-7 cancer cells and of micellar BTZ/17-AAG in MCF-7, PC3, MDA-MB-231 and HepG2 cells. PMID:22681126

  20. Effects of 17-AAG on the cell cycle and apoptosis of H446 cells and the associated mechanisms

    PubMed Central

    Zhao, Xuerong; Wang, Jianping; Xiao, Lijun; Xu, Qian; Zhao, Enhong; Zheng, Xin; Zheng, Huachuan; Zhao, Shuang; Ding, Shi

    2016-01-01

    As a heat shock protein 90 inhibitor, 17-allyl-amino-17-demethoxygeldanamycin (17-AAG) has been studied in numerous types of cancer, however the effects of 17-AAG on apoptosis and the cell cycle of H446 cells remain unclear. In the current study, the MTT method was used to evaluate the inhibitory effects of different durations and doses of 17-AAG treatment on the proliferation of H446 cells. The cells were stained with Annexin-fluorescein isothiocyanate/propidium iodide and measured by flow cytometry, and the gene and protein expression levels of signal transducer and activator of transcription 3 (STAT3), survivin, cyclin D1, cyt-C, caspase 9 and caspase 3 were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The results indicated that with treatment with 1.25–20 mg/l 17-AAG for 24 and 48 h, significant inhibition of H446 cell proliferation was observed in a time- and dose-dependent manner. With treatment of 3.125, 6.25 and 12.5 mg/l 17-AAG for 48 h, significant apoptosis and cell cycle arrest was observed. The results indicated that the gene and protein expression levels of STAT3, survivin and cyclin D1 were downregulated, and cyt-C, caspase 9 and caspase 3 were upregulated by 17-AAG in a dose-dependent manner when the cells were treated with 3.125 and 6.25 mg/l 17-AAG for 48 h. The results indicated that 17-AAG is able to inhibit the cell proliferation, induce apoptosis and G2/M arrest and downregulate the gene and protein expression levels of STAT3, survivin and cyclin D1, and upregulate gene and protein expression of cyt-C, caspase 9, caspase 3. PMID:27279418

  1. 17AAG improves histological and functional outcomes in a rat CCI model through autophagy activation and apoptosis attenuation.

    PubMed

    Ma, Lixin; Li, Zefu; Liu, Zhihui; Li, Meng; Sui, Dehua; Liu, Yongliang; Shao, Wei; Wang, Bo; Liu, Pengfei; Li, Gang

    2015-07-10

    Traumatic brain injury (TBI) is caused by both primary and secondary injury mechanisms, all of which cause neuronal cell death and functional deficits. Both apoptosis and autophagy participated in neuronal cell death and functional loss induced following TBI. Preclinical findings implicate that 17-allylamino-demethoxygeldanamycin (17-AAG), an anticancer drug in clinical, present neuroprotection actions in multiple neurological disorders, but whether 17-AAG is capable of modulating neuronal autophagy has never been addressed. The present study was designed to determine the hypothesis that17-AAG treatment could confer neuroprotection in a rat model of TBI. We also used an autophagy inhibitor 3-methyladenine (3-MA) as well as an autophagy inducer rapamycin (RAPA) to test its underlining mechanisms. Our results showed that post-TBI administration of 17-AAG could attenuate brain edema, decrease neuronal death, as well as improve the recovery of motor function. Afterwards, in our model, 17-AAG treatment protected against TBI-induced apoptosis activation as well as enhanced neuronal autophagy. The present study provides novel clues in understanding the mechanisms of which 17-AAG exerts its neuroprotective activity on neurological disorders. PMID:25957556

  2. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG.

    PubMed

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  3. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

    PubMed Central

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  4. The HSP90 inhibitor, 17AAG, protects the intestinal stem cell niche and inhibits graft versus host disease development.

    PubMed

    Joly, A-L; Deepti, A; Seignez, A; Goloudina, A; Hebrard, S; Schmitt, E; Richaud, S; Fourmaux, E; Hammann, A; Collura, A; Svrcek, M; Jego, G; Robinet, E; Solary, E; Demidov, O; Kohli, E; Garrido, C

    2016-06-01

    Graft versus host disease (GvHD), which is the primary complication of allogeneic bone marrow transplantation, can alter the intestinal barrier targeted by activated donor T-cells. Chemical inhibition of the stress protein HSP90 was demonstrated in vitro to inhibit T-cell activation and to modulate endoplasmic reticulum (ER) stress to which intestinal cells are highly susceptible. Since the HSP90 inhibitor 17-allylamino-demethoxygeldanamycin (17AAG) is developed in clinics, we explored here its ability to control intestinal acute GvHD in vivo in two mouse GvHD models (C57BL/6BALB/c and FVB/NLgr5-eGFP), ex vivo in intestine organoids and in vitro in intestinal epithelial cultures. We show that 17AAG decreases GvHD-associated mortality without impairing graft versus leukemia effect. While 17AAG effect in T-cell activation is just moderate at the dose used in vivo, we observe a striking intestinal integrity protection. At the intestine level, the drug promotes the splicing of the transcription factor X-box binding protein 1 (XBP1), which is a key component of the ER stress. This effect is associated with a decrease in intestinal damage and an increase in Lgr5(+) stem cells, Paneth cells and defensins production. The importance of XBP1 splicing control is further confirmed in cultured cells and organoids of primary intestinal epithelium where XBP1 is either shRNA depleted or inhibited with toyocamycin. In conclusion, 17AAG has a protective effect on the epithelial intestinal barrier in mouse models of acute GvHD. This compound deserves to be tested in the therapeutic control of acute GvHD. PMID:26364610

  5. Synthesis of Reblastatin, Autolytimycin, Non-Benzoquinone Analogs: Potent Inhibitors of Heat Shock Protein 90 (Hsp90)

    PubMed Central

    Wrona, Iwona E.; Gozman, Alexander; Taldone, Tony; Chiosis, Gabriela; Panek, James S.

    2010-01-01

    A full account of an asymmetric synthesis of reblastatin (1), the first total synthesis of autolytimycin (2) and related structural compounds is described. The syntheses expand the utility of a highly regio-and diastereoselective hydrometalation aldehyde addition sequence to assemble the fully functionalized ansa chain of the natural products. Also documented is an intramolecular copper-mediated amidation reaction to close the 19-membered macrolactams. The amidation reaction was also employed for the generation of structural derivatives (6–9) of phenolic ansamycins. Ansamycin natural products and selected structural analogs were evaluated in a competitive binding assay to breast cancer cell lysate and a cytotoxicity assay. Both reblastatin (1) and autolytimycin (2) were shown to bind the Hsp90 protein with enhanced binding activity (~25 nM) than 17-allylamino-17-demethoxygeldanamycin (17-AAG, 4), a geldanamycin (3) derivative currently under evaluation for treatment of cancer (~100 nM). PMID:20392070

  6. Preparation and evaluation of 17-allyamino-17-demethoxygeldanamycin (17-AAG)-loaded poly(lactic acid-co-glycolic acid) nanoparticles.

    PubMed

    Pradhan, Roshan; Poudel, Bijay Kumar; Choi, Ju Yeon; Choi, Im Soon; Shin, Beom Soo; Choi, Han-Gon; Yong, Chul Soon; Kim, Jong Oh

    2015-01-01

    In the present study, we developed the novel 17-allyamino-17-demethoxygeldanamycin (17-AAG)-loaded poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles (NPs) using the combination of sodium lauryl sulfate and poloxamer 407 as the anionic and non-ionic surfactant for stabilization. The PLGA NPs were prepared by emulsification/solvent evaporation method. Both the drug/polymer ratio and phase ratio were 1:10 (w/w). The optimized formulation of 17-AAG-loaded PLGA NPs had a particle size and polydispersity index of 151.6 ± 2.0 and 0.152 ± 0.010 nm, respectively, which was further supported by TEM image. The encapsulation efficiency and drug loading capacity were 69.9 and 7.0%, respectively. In vitro release study showed sustained release. When in vitro release data were fitted to Korsmeyer-Peppas model, the n value was 0.468, which suggested that the drug was released by anomalous or non-Fickian diffusion. In addition, 17-AAG-loaded PLGA NPs in 72 h, displayed approximately 60% cell viability reduction at 10 µg/ml 17-AAG concentration, in MCF-7 cell lines, indicating sustained release from NPs. Therefore, our results demonstrated that incorporation of 17-AAG into PLGA NPs could provide a novel effective nanocarrier for the treatment of cancer. PMID:24824337

  7. Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells.

    PubMed

    Weng, Shao-Hsing; Tseng, Sheng-Chieh; Huang, Yu-Ching; Chen, Huang-Jen; Lin, Yun-Wei

    2012-07-01

    Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1-1 μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells. PMID:22480737

  8. Identification of the Plant Compound Geraniin as a Novel Hsp90 Inhibitor

    PubMed Central

    Vassallo, Antonio; Vaccaro, Maria Carmela; De Tommasi, Nunziatina; Dal Piaz, Fabrizio; Leone, Antonella

    2013-01-01

    Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90) binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino)-17-demethoxygeldanamycin (17AGG). Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose−dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down−regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors. PMID:24066128

  9. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β

    PubMed Central

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  10. Critical regulation of TGFbeta signaling by Hsp90.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

    2008-07-01

    Transforming growth factor beta (TGFbeta) controls a diverse set of cellular processes by activating TGFbeta type I (TbetaRI) and type II (TbetaRII) serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated by Smad2 and Smad3, which are phosphorylated in their SXS motif by activated TbetaRI. The 90-kDa heat-shock protein (Hsp90) is a molecular chaperone facilitating the folding and stabilization of many protein kinases and intracellular signaling molecules. Here, we present evidence identifying a critical role for Hsp90 in TGFbeta signaling. Inhibition of Hsp90 function by using small-molecule inhibitors such as 17-allylamino-17-demethoxygeldanamycin (17AAG), and also at the genetic level, blocks TGFbeta-induced signaling and transcriptional responses. Furthermore, we identify TbetaRI and TbetaRII as Hsp90-interacting proteins in vitro and in vivo and demonstrate that inhibition of Hsp90 function increases TbetaR ubiquitination and degradation dependent on the Smurf2 ubiquitin E3 ligase. Our data reveal an essential level of TGFbeta signaling regulation mediated by Hsp90 by its ability to chaperone TbetaRs and also implicate the use of Hsp90 inhibitors in blocking undesired activation of TGFbeta signaling in diseases. PMID:18591668

  11. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

    PubMed

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  12. Disruption of the EF-2 kinase/Hsp90 protein complex: a possible mechanism to inhibit glioblastoma by geldanamycin.

    PubMed

    Yang, J; Yang, J M; Iannone, M; Shih, W J; Lin, Y; Hait, W N

    2001-05-15

    Glioblastoma multiforme is the most treatment-resistant brain tumor. Elongation factor-2 (EF-2) kinase (calmodulin kinase III) is a unique protein kinase that is overexpressed in glioma cell lines and in human surgical specimens. Several mitogens activate this kinase and inhibitors block mitogen activation and produce cell death. Geldanamycin (GA) is a benzoquinone ansamycin antibiotic that disrupts Hsp90-protein interactions. Because EF-2 kinase is chaperoned by Hsp90, we investigated the effects of GA on the viability of glioma cells, the expression of EF-2 kinase protein, and the interaction between Hsp90 and EF-2 kinase. GA was a potent inhibitor of the clonogenicity of four glioma cells lines with IC(50)s ranging from 1 to 3 nM. 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic and less potent derivative of GA, inhibited the clonogenicity of glioma cells with IC(50) values of 13 nM in C6 cells and 35 nM in T98G cells. Treatment of cell lines for 24-48 h of GA or 17-AAG disrupted EF-2-kinase/Hsp90 interactions as measured by coimmunoprecipitation, resulting in a decreased amount of recoverable kinase in cell lysates. The ability of GA to inhibit the growth of glioma cells was abrogated by overexpressing EF-2 kinase. In addition, 17-AAG significantly inhibited the growth of a glioma xenograft in nude mice. These studies demonstrate for the first time the activity of GAs against human gliomas in vitro and in vivo and suggest that destruction of EF-2 kinase may be an important cytotoxic mechanism of this unique class of drug. PMID:11358819

  13. Inhibition of Heat Shock Protein 90 Prevents HIV Rebound.

    PubMed

    Joshi, Pheroze; Maidji, Ekaterina; Stoddart, Cheryl A

    2016-05-01

    HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4(+) T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ(-/-) bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs. PMID:26957545

  14. Inhibition of Heat Shock Protein 90 Prevents HIV Rebound*

    PubMed Central

    Joshi, Pheroze; Maidji, Ekaterina; Stoddart, Cheryl A.

    2016-01-01

    HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4+ T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ−/− bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs. PMID:26957545

  15. Up-regulation of Gadd45α after Exposure to Metal Nanoparticles: the Role of Hypoxia Inducible Factor 1α

    PubMed Central

    Feng, Lingfang; Zhang, Yue; Jiang, Mizu; Mo, Yiqun; Wan, Rong; Jia, Zhenyu; Tollerud, David J.; Zhang, Xing; Zhang, Qunwei

    2014-01-01

    The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as Cobalt and Titanium dioxide (Nano-Co and Nano-TiO2) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage and apoptosis. The growth arrest and DNA damage-inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non-toxic doses of Nano-Co and Nano-TiO2 were selected to treat cells. Our results showed that Nano-Co caused a dose- and time-dependent increase in Gadd45α expression, but Nano-TiO2 did not. To investigate the potential pathways involved in Nano-Co-induced Gadd45α up-regulation, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in PW cells exposed to Nano-Co and Nano-TiO2. Our results showed that exposure to Nano-Co caused HIF-1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock-out cells [HIF-1α (−/−)] and its wild-type cells [HIF-1α (+/+)] were used. Our results demonstrated that Nano-Co caused a dose- and time-dependent increase in Gadd45α expression in wild-type HIF-1α (+/+) cells, but only a slight increase in HIF-1α (−/−) cells. Pre-treatment of PW cells with heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Co significantly abolished the Nano-Co-induced Gadd45α expression. These results suggest that HIF-1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano-Co. These findings may have important implications for understanding the potential health

  16. A novel temperature-responsive micelle for enhancing combination therapy

    PubMed Central

    Peng, Cheng-Liang; Chen, Yuan-I; Liu, Hung-Jen; Lee, Pei-Chi; Luo, Tsai-Yueh; Shieh, Ming-Jium

    2016-01-01

    A novel thermosensitive polymer p(N-isopropylacrylamide-co-poly[ethylene glycol] methyl ether acrylate)-block-poly(epsilon-caprolactone), p(NIPAAM-co-PEGMEA)-b-PCL, was synthesized and developed as nanomicelles. The hydrophobic heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin and the photosensitizer cyanine dye infrared-780 were loaded into the core of the micelles to achieve both chemotherapy and photothermal therapy simultaneously at the tumor site. The release of the drug could be controlled by varying the temperature due to the thermosensitive nature of the micelles. The micelles were less than 200 nm in size, and the drug encapsulation efficiency was >50%. The critical micelle concentrations were small enough to allow micelle stability upon dilution. Data from cell viability and animal experiments indicate that this combination treatment using photothermal therapy with chemotherapy had synergistic effects while decreasing side effects. PMID:27524894

  17. A novel temperature-responsive micelle for enhancing combination therapy.

    PubMed

    Peng, Cheng-Liang; Chen, Yuan-I; Liu, Hung-Jen; Lee, Pei-Chi; Luo, Tsai-Yueh; Shieh, Ming-Jium

    2016-01-01

    A novel thermosensitive polymer p(N-isopropylacrylamide-co-poly[ethylene glycol] methyl ether acrylate)-block-poly(epsilon-caprolactone), p(NIPAAM-co-PEGMEA)-b-PCL, was synthesized and developed as nanomicelles. The hydrophobic heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin and the photosensitizer cyanine dye infrared-780 were loaded into the core of the micelles to achieve both chemotherapy and photothermal therapy simultaneously at the tumor site. The release of the drug could be controlled by varying the temperature due to the thermosensitive nature of the micelles. The micelles were less than 200 nm in size, and the drug encapsulation efficiency was >50%. The critical micelle concentrations were small enough to allow micelle stability upon dilution. Data from cell viability and animal experiments indicate that this combination treatment using photothermal therapy with chemotherapy had synergistic effects while decreasing side effects. PMID:27524894

  18. Tanespimycin and Bortezomib in Treating Patients With Advanced Solid Tumors or Lymphomas

    ClinicalTrials.gov

    2014-02-21

    Adult Grade III Lymphomatoid Granulomatosis; AIDS-related Peripheral/Systemic Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Nodal Marginal Zone B-cell Lymphoma; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult T-cell Leukemia/Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Stage III Adult Burkitt Lymphoma; Stage III Adult Diffuse Large Cell Lymphoma; Stage III Adult Diffuse Mixed Cell Lymphoma; Stage III Adult Diffuse Small Cleaved Cell Lymphoma; Stage III Adult Hodgkin Lymphoma; Stage III Adult Immunoblastic Large Cell Lymphoma; Stage III Adult Lymphoblastic Lymphoma; Stage III Adult T-cell Leukemia/Lymphoma; Stage III Cutaneous T-cell Non-Hodgkin Lymphoma; Stage III Grade 1 Follicular Lymphoma; Stage III Grade 2 Follicular Lymphoma; Stage III Grade 3 Follicular Lymphoma; Stage III Mantle Cell Lymphoma; Stage III Marginal Zone Lymphoma; Stage III Mycosis Fungoides/Sezary Syndrome; Stage III Small Lymphocytic Lymphoma; Stage IV Adult Burkitt Lymphoma; Stage IV Adult Diffuse Large Cell Lymphoma; Stage IV Adult Diffuse Mixed Cell Lymphoma; Stage IV Adult Diffuse Small Cleaved Cell Lymphoma; Stage IV Adult Hodgkin Lymphoma; Stage IV Adult Immunoblastic Large Cell Lymphoma; Stage IV Adult Lymphoblastic Lymphoma; Stage IV Adult T-cell Leukemia/Lymphoma; Stage IV Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IV Grade 1 Follicular Lymphoma; Stage IV Grade 2 Follicular Lymphoma; Stage IV Grade 3 Follicular Lymphoma; Stage IV Mantle Cell Lymphoma; Stage IV Marginal Zone Lymphoma; Stage IV Mycosis Fungoides/Sezary Syndrome; Stage IV Small Lymphocytic Lymphoma; Unspecified Adult Solid Tumor, Protocol Specific; Waldenström Macroglobulinemia

  19. Tanespimycin in Treating Young Patients With Recurrent or Refractory Leukemia or Solid Tumors

    ClinicalTrials.gov

    2013-06-03

    Childhood Chronic Myelogenous Leukemia; Childhood Desmoplastic Small Round Cell Tumor; Disseminated Neuroblastoma; Metastatic Childhood Soft Tissue Sarcoma; Metastatic Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor; Metastatic Osteosarcoma; Previously Treated Childhood Rhabdomyosarcoma; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Childhood Rhabdomyosarcoma; Recurrent Childhood Soft Tissue Sarcoma; Recurrent Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor; Recurrent Neuroblastoma; Recurrent Osteosarcoma

  20. Tanespimycin and Cytarabine in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, Chronic Myelomonocytic Leukemia, or Myelodysplastic Syndromes

    ClinicalTrials.gov

    2013-09-27

    Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Basophilic Leukemia; Adult Acute Eosinophilic Leukemia; Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Blastic Phase Chronic Myelogenous Leukemia; Chronic Myelomonocytic Leukemia; de Novo Myelodysplastic Syndromes; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts in Transformation; Relapsing Chronic Myelogenous Leukemia; Secondary Acute Myeloid Leukemia; Secondary Myelodysplastic Syndromes

  1. HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

    PubMed

    van der Kraan, A Gabrielle J; Chai, Ryan C C; Singh, Preetinder P; Lang, Benjamin J; Xu, Jiake; Gillespie, Matthew T; Price, John T; Quinn, Julian M W

    2013-04-15

    The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFβ (transforming growth factor β) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity. PMID:23379601

  2. Inhibition of heat-shock protein 90 sensitizes liver cancer stem-like cells to magnetic hyperthermia and enhances anti-tumor effect on hepatocellular carcinoma-burdened nude mice

    PubMed Central

    Yang, Rui; Tang, Qiusha; Miao, Fengqin; An, Yanli; Li, Mengfei; Han, Yong; Wang, Xihui; Wang, Juan; Liu, Peidang; Chen, Rong

    2015-01-01

    Purpose To explore the thermoresistance and expression of heat-shock protein 90 (HSP90) in magnetic hyperthermia-treated human liver cancer stem-like cells (LCSCs) and the effects of a heat-shock protein HSP90 inhibitor 17-allylamino-17-demethoxgeldanamycin (17-AAG) on hepatocellular carcinoma-burdened nude mice. Methods CD90+ LCSCs were isolated by magnetic-activated cell sorting from BEL-7404. Spheroid formation, proliferation, differentiation, drug resistance, and tumor formation assays were performed to identify stem cell characteristics. CD90-targeted thermosensitive magnetoliposomes (TMs)-encapsulated 17-AAG (CD90@17-AAG/TMs) was prepared by reverse-phase evaporation and its characteristics were studied. Heat tolerance in CD90+ LCSCs and the effect of CD90@17-AAG/TMs-mediated heat sensitivity were examined in vitro and in vivo. Results CD90+ LCSCs showed significant stem cell-like properties. The 17-AAG/TMs were successfully prepared and were spherical in shape with an average size of 128.9±7.7 nm. When exposed to magnetic hyperthermia, HSP90 was up-regulated in CD90+ LCSCs. CD90@17-AAG/TMs inhibited the activity of HSP90 and increased the sensitivity of CD90+ LCSCs to magnetic hyperthermia. Conclusion The inhibition of HSP90 could sensitize CD90+ LCSCs to magnetic hyperthermia and enhance its anti-tumor effects in vitro and in vivo. PMID:26677324

  3. Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis.

    PubMed

    Grieve, Stacy; Gao, Yan; Hall, Christine; Hu, Jing; Greer, Peter A

    2016-08-01

    Calpain is an intracellular Ca(2+)-regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2(+) (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27(KIP1) and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics. PMID:27215381

  4. Radiosensitization of Human Vascular Endothelial Cells Through Hsp90 Inhibition With 17-N-Allilamino-17-Demethoxygeldanamycin

    SciTech Connect

    Kabakov, Alexander E. Makarova, Yulia M.; Malyutina, Yana V.

    2008-07-01

    Purpose: In addition to invasive tumor cells, endothelial cells (ECs) of the tumor vasculature are an important target for anticancer radiotherapy. The purpose of the present work is to investigate how 17-N-allilamino-17-demethoxygeldanamycin (17AAG), known as an anticancer drug inhibiting heat shock protein 90 (Hsp90), modifies radiation responses of human vascular ECs. Methods and Materials: The ECs cultured from human umbilical veins were exposed to {gamma}-irradiation, whereas some EC samples were pretreated with growth factors and/or 17AAG. Postirradiation cell death/survival and morphogenesis were assessed by means of terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling or annexin V staining and clonogenic and tube-formation assays. The 17AAG-affected expression and phosphorylation of radioresistance-related proteins were probed by means of immunoblotting. Dominant negative or constitutively activated Akt was transiently expressed in ECs to manipulate Akt activity. Results: It was found that nanomolar concentrations of 17AAG sensitize ECs to relatively low doses (2-6 Gy) of {gamma}-irradiation and abolish the radioprotective effects of vascular endothelial growth factor and basic fibroblast growth factor. The drug-induced radiosensitization of ECs seems to be caused by prevention of Hsp90-dependent phosphorylation (activation) of Akt that results in blocking the radioprotective phosphatidylinositol 3-kinase/Akt pathway. Conclusions: Clinically achievable concentrations of 17AAG can decrease the radioresistance intrinsic to vascular ECs and minimize the radioprotection conferred upon them by tumor-derived growth factors. These findings characterize 17AAG as a promising radiosensitizer for the tumor vasculature.

  5. Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer.

    PubMed

    Ma, Liang; Sato, Fuminori; Sato, Ryuta; Matsubara, Takanori; Hirai, Kenichi; Yamasaki, Mutsushi; Shin, Toshitaka; Shimada, Tatsuo; Nomura, Takeo; Mori, Kenichi; Sumino, Yasuhiro; Mimata, Hiromitsu

    2014-06-01

    Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer. PMID:24718854

  6. Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer

    PubMed Central

    MA, LIANG; SATO, FUMINORI; SATO, RYUTA; MATSUBARA, TAKANORI; HIRAI, KENICHI; YAMASAKI, MUTSUSHI; SHIN, TOSHITAKA; SHIMADA, TATSUO; NOMURA, TAKEO; MORI, KENICHI; SUMINO, YASUHIRO; MIMATA, HIROMITSU

    2014-01-01

    Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer. PMID:24718854

  7. Targeting GRP75 Improves HSP90 Inhibitor Efficacy by Enhancing p53-Mediated Apoptosis in Hepatocellular Carcinoma

    PubMed Central

    Yang, Ling; Liu, Xiaoyu; E, Qiukai; Gao, Peiye; Ye, Xiaofei; Liu, Wen; Zuo, Ji

    2014-01-01

    Heat shock protein 90 (HSP90) inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70) family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs. PMID:24465691

  8. The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

    PubMed Central

    Shi, Chao; Huang, Xuan; Zhang, Bin; Zhu, Dan; Luo, Huqiao; Lu, Quqin; Xiong, Wen-Cheng; Mei, Lin; Luo, Shiwen

    2015-01-01

    Background Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear. Results In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells. Conclusions Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD. PMID:26414348

  9. HSP90 Inhibition Suppresses PGE2 Production via Modulating COX-2 and 15-PGDH Expression in HT-29 Colorectal Cancer Cells.

    PubMed

    Mohammadi, A; Yaghoobi, M M; Gholamhoseinian Najar, A; Kalantari-Khandani, B; Sharifi, H; Saravani, M

    2016-06-01

    The existence of multiple-interactive roles between several signaling pathways in tumorigenesis shows the significance of pharmacological factors like heat shock protein 90 (HSP90) inhibitors which control several signaling pathways simultaneously. HSP90 as a molecular chaperone supports the active conformational structure and function of several oncogenic signal proteins, termed "client" proteins, some of them act as a link between cancer and inflammation. Prostaglandin E2 (PGE2) is one of the major mediators of inflammation in colorectal cancer development and progress. However, the relationship between chaperone activity of HSP90 and PGE2 levels remains unclear. We evaluated the inhibitory effects of 17-demethoxy-17-allylamino geldanamycin (1 7-AAG), an HSP90 inhibitor, on PGE2 levels in HT-29 colorectal cancer cells. For the first time, we showed inhibitory effects of 17-AAG, on PGE2 levels in HT-29 colorectal cancer cells. 17-AAG inhibited PMA-induced cyclooxygenase-2 (COX-2) mRNA expression and protein level. We showed 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression induced by 17-AAG treatment at both mRNA and protein levels. In conclusion, we found that inhibitory effects of 17-AAG on PGE2 levels in HT-29 colorectal cancer cells were mediated through modulating COX-2 and 15-PGDH expression. PMID:27075590

  10. BIIB021, a synthetic Hsp90 inhibitor, has broad application against tumors with acquired multidrug resistance.

    PubMed

    Zhang, Hong; Neely, Laura; Lundgren, Karen; Yang, Yong-Ching; Lough, Rachel; Timple, Noel; Burrows, Francis

    2010-03-01

    17-AAG, the first-generation clinical Hsp90 inhibitor, exhibits promising antitumor activity in clinical studies, but is limited by poor solubility and hepatotoxicity. To pursue compounds with better biopharmaceutical properties, we have developed a series of fully synthetic orally bioavailable inhibitors of Hsp90. Here, we report that 17-AAG and other ansamycin derivatives are inactive in P-gp and/or MRP-1 expressing cell lines and sensitivity could be restored by coadministration of P-gp or MRP inhibitors. In contrast, the synthetic Hsp90 inhibitor, BIIB021 was active in these models. Accordingly, BIIB021 was considerably more active than 17-AAG against adrenocortical carcinoma, a tumor that naturally expresses P-gp, both in vitro and in vivo. This efflux pump-mediated resistance is manifested in both cytotoxicity assays and measurements of target inhibition, such as client protein degradation. Other than this, the cytotoxic activity of BIIB021 was also not influenced by loss of NQO1 or Bcl-2 overexpression, molecular lesions that do not prevent client loss but are nonetheless associated with reduced cell killing by 17-AAG. Our results indicate that the activity of 17-AAG and other ansamycins may be curtailed in tumors that have upregulated efflux pumps or antiapoptotic proteins or other genetic alterations. These data indicate that the new generation of synthetic anti-Hsp90 drugs, exemplified by BIIB021 that is currently undergoing Phase II testing, may have broader application against tumors with acquired multidrug resistance or tumors located in organs protected by MDR proteins, such as the adrenal glands, brain and testis. PMID:19676042

  11. Multidrug PLA-PEG filomicelles for concurrent delivery of anticancer drugs-The influence of drug-drug and drug-polymer interactions on drug loading and release properties.

    PubMed

    Jelonek, Katarzyna; Li, Suming; Kaczmarczyk, Bożena; Marcinkowski, Andrzej; Orchel, Arkadiusz; Musiał-Kulik, Monika; Kasperczyk, Janusz

    2016-08-20

    This study aimed to analyze the influence of drug-drug and drug-polymer interactions on drug loading and release properties of multidrug micelles. Three hydrophobic drugs-paclitaxel (Ptx), 17-AAG and rapamycin (Rap) were incorporated in poly(l-lactide)-poly(ethylene glycol) (PLA-PEG) filomicelles. Double loaded micelles containing Ptx and 17-AAG were used for the sake of comparison. (1)H NMR confirmed the effective incorporation of the various drugs in micelles, and HPLC allowed to determine the drug loading contents. FTIR was used to evaluate interactions between particular drugs and between drugs and copolymer. Ptx and 17-AAG present similar loading efficiencies in double loaded micelles probably due to interactions of drugs with each other and also with the copolymer. In contrast, unequal drug loading properties are observed for triple loaded micelles. Rapamycin shows very weak interactions with the copolymer, and displays the lowest loading efficiency. In vitro release of drugs from micelles was realized in pH 7.4 phosphate buffered saline at 37°C, and monitored by HPLC. Similar release profiles are observed for the three drugs: a strong burst followed by slower release. Nevertheless, Ptx release from micelles is significantly slower as compared to 17-AAG and Rap, probably due to interactions of NH and OH groups of Ptx with the carbonyl group of PLA. In vitro cytotoxicity of Ptx/17-AAG/Rap loaded micelles and a mixture of free drugs was determined. Drug loaded micelles exhibit advantageous effect of prolonged drug release and cytotoxic activity against Caco-2 cells, which makes them a promising solution for simultaneous drug delivery to solid tumors. Therefore, understanding of interactions within multidrug micelles should be a valuable approach for the development of concurrent delivery systems of anticancer drugs with tailored properties. PMID:27346726

  12. Identification of epipolythiodioxopiperazines HDN-1 and chaetocin as novel inhibitor of heat shock protein 90

    PubMed Central

    Song, Xiaoping; Zhao, Zhimin; Qi, Xin; Tang, Shuai; Wang, Qiang; Zhu, Tianjiao; Gu, Qianqun; Liu, Ming; Li, Jing

    2015-01-01

    The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an important target for cancer treatment. HDN-1, an epipolythiopiperazine-2, 5-diones (ETPs) compound, was here identified as a new Hsp90 inhibitor. HDN-1 bound directly to C-terminus of Hsp90α, resulting in a potential conformational change that interfered with the binding of 17-AAG and novobiocin to Hsp90α. In contrast, association of 17-AAG, novobiocin or ATP with Hsp90α did not prevent the binding HDN-1 to Hsp90α. HDN-1 in combination with 17-AAG exhibited an enhanced inhibitory effect on non-small lung cancer cell proliferation. Molecular docking analyses revealed that HDN-1 bound to Hsp90α at C-terminal 526–570 region. In addition, HDN-1 degraded multiple oncoproteins and promoted EGF-induced wild type and mutated EGFR downregulation. Notably, chaetocin, used as a SUV39H1 inhibitor with similar structure to HDN-1, bound to Hsp90 and degraded Hsp90 client proteins and SUV39H1 as did HDN-1. These results indicate that HDN-1 and chaetocin are inhibitors of Hsp90 and that SUV39H1 is a novel client protein of Hsp90. PMID:25742791

  13. Dual mode of cancer cell destruction for pancreatic cancer therapy using Hsp90 inhibitor loaded polymeric nano magnetic formulation.

    PubMed

    Rochani, Ankit K; Balasubramanian, Sivakumar; Ravindran Girija, Aswathy; Raveendran, Sreejith; Borah, Ankita; Nagaoka, Yutaka; Nakajima, Yoshikata; Maekawa, Toru; Kumar, D Sakthi

    2016-09-10

    Heat Shock Protein 90 (Hsp90) has been extensively explored as a potential drug target for cancer therapies. 17- N-allylamino- 17-demethoxygeldanamycin (17AAG) was the first Hsp90 inhibitor to enter clinical trials for cancer therapy. However, native drug is being shown to have considerable anticancer efficacy against pancreatic cancer when used in combination therapy regime. Further, magnetic hyperthermia has shown to have promising effects against pancreatic cancer in combination with known cyto-toxic drugs under both target and non-targeted scenarios. Hence, in order to enhance the efficacy of 17AAG against pancreatic cancer, we developed poly (lactic-co-glycolic acid) (PLGA) coated, 17AAG and Fe3O4 loaded magnetic nanoparticle formulations by varying the relative concentration of polymer. We found that polymer concentration affects the magnetic strength and physicochemical properties of formulation. We were also able to see that our aqueous dispensable formulations were able to provide anti-pancreatic cancer activity for MIA PaCa-2 cell line in dose and time dependent manner in comparison to mice fibroblast cell lines (L929). Moreover, the in-vitro magnetic hyperthermia against MIA PaCa-2 provided proof principle that our 2-in-1 particles may work against cancer cell lines effectively. PMID:27469073

  14. A rat retinal damage model predicts for potential clinical visual disturbances induced by Hsp90 inhibitors

    SciTech Connect

    Zhou, Dan; Liu, Yuan; Ye, Josephine; Ying, Weiwen; Ogawa, Luisa Shin; Inoue, Takayo; Tatsuta, Noriaki; Wada, Yumiko; Koya, Keizo; Huang, Qin; Bates, Richard C.; Sonderfan, Andrew J.

    2013-12-01

    In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24 h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6 h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6 h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential. - Highlights: • In human trials some Hsp90 inhibitors cause visual disorders, others do not. • Prolonged inhibition of Hsp90 in the rat eye results in photoreceptor cell death. • Retina/plasma ratio and retinal

  15. Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo.

    PubMed

    Shammas, M A; Koley, H; Bertheau, R C; Neri, P; Fulciniti, M; Tassone, P; Blotta, S; Protopopov, A; Mitsiades, C; Batchu, R B; Anderson, K C; Chin, A; Gryaznov, S; Munshi, N C

    2008-07-01

    Human telomerase, the reverse transcriptase which extends the life span of a cell by adding telomeric repeats to chromosome ends, is expressed in most cancer cells but not in the majority of normal somatic cells. Inhibition of telomerase therefore holds great promise as anticancer therapy. We have synthesized a novel telomerase inhibitor GRN163L, a lipid-attached phosphoramidate oligonucleotide complementary to template region of the RNA subunit of telomerase. Here, we report that GRN163L is efficiently taken up by human myeloma cells without any need of transfection and is resistant to nucleolytic degradation. The exposure of myeloma cells to GRN163L led to an effective inhibition of telomerase activity, reduction of telomere length and apoptotic cell death after a lag period of 2-3 weeks. Mismatch control oligonucleotides had no effect on growth of myeloma cells. The in vivo efficacy of GRN163L was confirmed in two murine models of human multiple myeloma. In three independent experiments, significant reduction in tumor cell growth and better survival than control mice was observed. Furthermore, GRN163L-induced myeloma cell death could be significantly enhanced by Hsp90 inhibitor 17AAG. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with 17AAG. PMID:18449204

  16. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    SciTech Connect

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  17. Identifying Inhibitors of Epithelial-Mesenchymal Transition by Connectivity-Map Based Systems Approach

    PubMed Central

    Reka, Ajaya Kumar; Kuick, Rork; Kurapati, Himabindu; Standiford, Theodore J.; Omenn, Gilbert S.; Keshamouni, Venkateshwar G.

    2011-01-01

    Background Acquisition of mesenchymal phenotype by epithelial cells by means of epithelial mesenchymal transition (EMT) is considered as an early event in the multi-step process of tumor metastasis. Therefore, inhibition of EMT might be a rational strategy to prevent metastasis. Methods Utilizing the global gene expression profile from a cell culture model of TGF-β-induced EMT, we identified potential EMT inhibitors. We used a publicly available database (www.broad.mit.edu/cmap) comprising gene expression profiles obtained from multiple different cell lines in response to various drugs to derive negative correlations to EMT gene expression profile using Connectivity Map (C-Map), a pattern matching tool. Results Experimental validation of the identified compounds showed rapamycin as a novel inhibitor of TGF-β signaling along with 17-AAG, a known modulator of TGF-β pathway. Both of these compounds completely blocked EMT and the associated migratory and invasive phenotype. The other identified compound, LY294002, demonstrated a selective inhibition of mesenchymal markers, cell migration and invasion, without affecting the loss of E-cadherin expression or Smad phosphorylation. Conclusions Collectively, our data reveals that rapamycin is a novel modulator of TGF-β signaling, and along with 17-AAG and LY294002, could be used as therapeutic agent for inhibiting EMT. Also, this analysis demonstrates the potential of a systems approach in identifying novel modulators of a complex biological process. PMID:21964532

  18. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1.

    PubMed

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-11-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  19. Chemically Accessible Hsp90 Inhibitor That Does Not Induce a Heat Shock Response

    PubMed Central

    2014-01-01

    Recent cancer therapies have focused on targeting biology networks through a single regulatory protein. Heat shock protein 90 (hsp90) is an ideal oncogenic target as it regulates over 400 client proteins and cochaperones. However, clinical inhibitors of hsp90 have had limited success; the primary reason being that they induce a heat shock response. We describe the synthesis and biological evaluation of a new hsp90 inhibitor, SM253. The previous generation on which SM253 is based (SM145) has poor overall synthetic yields, low solubility, and micromolar cytotoxicity. By comparison SM253 has relatively high overall yields, good aqueous solubility, and is more cytotoxic than its parent compound. Verification that hsp90 is SM253’s target was accomplished using pull-down and protein folding assays. SM253 is superior to both SM145 and the clinical candidate 17-AAG as it decreases proteins related to the heat shock response by 2-fold, versus a 2–4-fold increase observed when cells are treated with 17-AAG. PMID:25050163

  20. Potent Antitrypanosomal Activities of Heat Shock Protein 90 Inhibitors In Vitro and In Vivo

    PubMed Central

    Meyer, Kirsten J.; Shapiro, Theresa A.

    2013-01-01

    African sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is universally fatal if untreated, and current drugs are limited by severe toxicities and difficult administration. New antitrypanosomals are greatly needed. Heat shock protein 90 (Hsp90) is a conserved and ubiquitously expressed molecular chaperone essential for stress responses and cellular signaling. We investigated Hsp90 inhibitors for their antitrypanosomal activity. Geldanamycin and radicicol had nanomolar potency in vitro against bloodstream-form T. brucei; novobiocin had micromolar activity. In structure-activity studies of geldanamycin analogs, 17-AAG and 17-DMAG were most selective against T. brucei as compared to mammalian cells. 17-AAG treatment sensitized trypanosomes to heat shock and caused severe morphological abnormalities and cell cycle disruption. Both oral and parenteral 17-DMAG cured mice of a normally lethal infection of T. brucei. These promising results support the use of inhibitors to study Hsp90 function in trypanosomes and to expand current clinical development of Hsp90 inhibitors to include T. brucei. PMID:23630365

  1. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    PubMed Central

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  2. Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma.

    PubMed

    Mimura, Naoya; Fulciniti, Mariateresa; Gorgun, Gullu; Tai, Yu-Tzu; Cirstea, Diana; Santo, Loredana; Hu, Yiguo; Fabre, Claire; Minami, Jiro; Ohguchi, Hiroto; Kiziltepe, Tanyel; Ikeda, Hiroshi; Kawano, Yutaka; French, Maureen; Blumenthal, Martina; Tam, Victor; Kertesz, Nathalie L; Malyankar, Uriel M; Hokenson, Mark; Pham, Tuan; Zeng, Qingping; Patterson, John B; Richardson, Paul G; Munshi, Nikhil C; Anderson, Kenneth C

    2012-06-14

    Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM. PMID:22538852

  3. In vitro discovery of promising anti-cancer drug combinations using iterative maximisation of a therapeutic index

    PubMed Central

    Kashif, M.; Andersson, C.; Hassan, S.; Karlsson, H.; Senkowski, W.; Fryknäs, M.; Nygren, P.; Larsson, R.; Gustafsson, M.G.

    2015-01-01

    In vitro-based search for promising anti-cancer drug combinations may provide important leads to improved cancer therapies. Currently there are no integrated computational-experimental methods specifically designed to search for combinations, maximizing a predefined therapeutic index (TI) defined in terms of appropriate model systems. Here, such a pipeline is presented allowing the search for optimal combinations among an arbitrary number of drugs while also taking experimental variability into account. The TI optimized is the cytotoxicity difference (in vitro) between a target model and an adverse side effect model. Focusing on colorectal carcinoma (CRC), the pipeline provided several combinations that are effective in six different CRC models with limited cytotoxicity in normal cell models. Herein we describe the identification of the combination (Trichostatin A, Afungin, 17-AAG) and present results from subsequent characterisations, including efficacy in primary cultures of tumour cells from CRC patients. We hypothesize that its effect derives from potentiation of the proteotoxic action of 17-AAG by Trichostatin A and Afungin. The discovered drug combinations against CRC are significant findings themselves and also indicate that the proposed strategy has great potential for suggesting drug combination treatments suitable for other cancer types as well as for other complex diseases. PMID:26392291

  4. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. PMID:22066472

  5. Hsp90 inhibitors reduce influenza virus replication in cell culture

    SciTech Connect

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin Brownlee, George

    2008-08-01

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

  6. Heat-shock protein dysregulation is associated with functional and pathological TDP-43 aggregation

    NASA Astrophysics Data System (ADS)

    Chang, Hsiang-Yu; Hou, Shin-Chen; Way, Tzong-Der; Wong, Chi-Huey; Wang, I.-Fan

    2013-11-01

    Conformational disorders are involved in various neurodegenerative diseases. Reactive oxygen species (ROS) are the major contributors to neurodegenerative disease; however, ROS that affect the structural changes in misfolded disease proteins have yet to be well characterized. Here we demonstrate that the intrinsic propensity of TDP-43 to aggregate drives the assembly of TDP-43-positive stress granules and soluble toxic TDP-43 oligomers in response to a ROS insult via a disulfide crosslinking-independent mechanism. Notably, ROS-induced TDP-43 protein assembly correlates with the dynamics of certain TDP-43-associated chaperones. The heat-shock protein (HSP)-90 inhibitor 17-AAG prevents ROS-induced TDP-43 aggregation, alters the type of TDP-43 multimers and reduces the severity of pathological TDP-43 inclusions. In summary, our study suggests that a common mechanism could be involved in the pathogenesis of conformational diseases that result from HSP dysregulation.

  7. Reversing drug resistance of cisplatin by hsp90 inhibitors in human ovarian cancer cells

    PubMed Central

    Zhang, Zhengmao; Xie, Zhen; Sun, Guangyu; Yang, Pingfang; Li, Jia; Yang, Hongfang; Xiao, Shuang; Liu, Yang; Qiu, Hongbing; Qin, Lijun; Zhang, Chao; Zhang, Fenghua; Shan, Baoen

    2015-01-01

    Objective: To investigate the mechanisms for reversing drug resistance of cisplatin (DDP) by Hsp90 inhibitors (geldanamycin (GA), 17-AAG, 17-DMAG) in human ovarian cancer. Methods: Cell proliferation rate in DDP resistant human ovarian cancer cell line SKOV3/DDP and its parent cell line SKOV3 after treatment with Hsp90 inhibitors and/or DDP were tested by MTT assay, and the reversing fold (RF) of DDP by Hsp90 inhibitors was calculated. Cell cycle and cell apoptosis status after treatment were analyzed by flow cytometry. The expression of multiple drug resistance related genes was analyzed by RT-PCR and Western-blot. Results: All three tested Hsp90 inhibitors synergistically inhibited the cell proliferation of SKOV3 with DDP and enhanced the sensitivity of SKOV3/DDP cells to DDP. The RF of DDP by Hsp90 inhibitors were all more than two fold. GA caused cell cycle arrest in G2/M phasein SKOV3 cells. 17-AAG increased cell apoptosis but did not change cell cycle in SKOV3/DDP cells. The mRNA and protein expression levels of various drug resistant related genes including LRP, GST-π, p53, bcl-2, survivin, ERCC1, XRCC1, BRCA1 and BRCA2 were more dramatically altered by Hsp90 inhibitors and DDP in combination compared to Hsp90 inhibitors or DDP treatment alone. Conclusions: Exposure of SKOV3/DDP cells to Hsp90 inhibitors and DDP in combination results in synergistic cytotoxic and pro-apoptotic effects. Hsp90 inhibitors reverse the drug resistance of SKOV3/DDP cells to DDP by modifying the expression of multiple drug resistance related genes. PMID:26221207

  8. Hsp90 inhibition protects against inherited retinal degeneration

    PubMed Central

    Aguilà, Mònica; Bevilacqua, Dalila; McCulley, Caroline; Schwarz, Nele; Athanasiou, Dimitra; Kanuga, Naheed; Novoselov, Sergey S.; Lange, Clemens A.K.; Ali, Robin R.; Bainbridge, James W.; Gias, Carlos; Coffey, Peter J.; Garriga, Pere; Cheetham, Michael E.

    2014-01-01

    The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1−/− cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function. PMID:24301679

  9. Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

    PubMed Central

    Tsou, Yueh-Liang; Lin, Yi-Wen; Chang, Hsuen-Wen; Lin, Hsiang-Yin; Shao, Hsiao-Yun; Yu, Shu-Ling; Liu, Chia-Chyi; Chitra, Ebenezer; Sia, Charles; Chow, Yen-Hung

    2013-01-01

    Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection. PMID:24098578

  10. Sensitization of Chemo-Resistant Human Chronic Myeloid Leukemia Stem-Like Cells to Hsp90 Inhibitor by SIRT1 Inhibition

    PubMed Central

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Kim, Mi-Ju; Hyun, Suh-Kyung; Gong, Eun-Ji; Oh, Won Keun; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies. PMID:26157347

  11. UVR Exposure Sensitizes Keratinocytes to DNA Adduct Formation

    PubMed Central

    Nair, Sudhir; Kekatpure, Vikram D.; Judson, Benjamin L.; Rifkind, Arleen B.; Granstein, Richard D.; Boyle, Jay O.; Subbaramaiah, Kotha; Guttenplan, Joseph B.; Dannenberg, Andrew J.

    2009-01-01

    Ultraviolet radiation (UVR) and exposure to tobacco smoke, a source of polycyclic aromatic hydrocarbons (PAH), have been linked to skin carcinogenesis. UVR-mediated activation of the aryl hydrocarbon receptor (AhR) stimulates the transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAH to genotoxic metabolites. We determined whether UVR exposure sensitized human keratinocytes to PAH-induced DNA adduct formation. UVR exposure induced CYP1A1 and CYP1B1 in HaCaT cells, an effect that was mimicked by photooxidized tryptophan (aTRP) and FICZ, a component of aTRP. UVR exposure or pretreatment with aTRP or FICZ also sensitized cells to benzo[a]pyrene (B[a]P) induced DNA adduct formation. α-Naphthoflavone (αNF), an AhR antagonist, suppressed UVR-, aTRP- and FICZ-mediated induction of CYP1A1 and CYP1B1 and inhibited B[a]P induced DNA adduct formation. Treatment with 17-AAG, a Hsp90 inhibitor, caused a marked decrease in levels of AhR, inhibited UVR-, aTRP- and FICZ-mediated induction of CYP1A1 and CYP1B1 and blocked the sensitization of HaCaT cells to B[a]P induced DNA adduct formation. FICZ has been suggested to be a physiological ligand of the AhR that may have systemic effects. Hence, studies of FICZ were also carried out in MSK-Leuk1 cells, a model of oral leukoplakia. Pretreatment with αNF or 17-AAG blocked FICZ-mediated induction of CYP1A1 and CYP1B1, and suppressed the increased B[a]P-induced DNA adduct formation. Collectively, these results suggest that sunlight may activate AhR signaling and thereby sensitize cells to PAH-mediated DNA adduct formation. Antagonists of AhR signaling may have a role in the chemoprevention of photocarcinogenesis. PMID:19789301

  12. Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

    PubMed Central

    Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

    2015-01-01

    Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and

  13. The potent activation of Ca(2+)-activated K(+) current by NVP-AUY922 in the human pancreatic duct cell line (PANC-1) possibly independent of heat shock protein 90 inhibition.

    PubMed

    Chiang, Nai-Jung; Wu, Sheng-Nan; Chen, Li-Tzong

    2015-04-01

    NVP-AUY922 (AUY) is a potent inhibitor of heat shock protein 90 (HSP90). Whether this compound can exert additional effects on membrane ion channels remains elusive. We investigated the effect of AUY on ion currents in human pancreatic duct epithelial cells (PDECs), including PANC-1 and MIA PaCa-2. AUY increased the amplitude of the K(+) current (IK) in PANC-1 cells shown by whole-cell configuration. Single-channel recordings revealed a large-conductance Ca(2+)-activated K(+) (BKCa) channel in PANC-1, but not in MIA PaCa-2. In cell-attached mode, AUY increased the probability of BKCa channel opening and also potentiated the activity of stretch-induced channels. However, other HSP inhibitors, 17-AAG or BIIB021 only slightly increased the activity of BKCa channels. In inside-out recordings, sodium hydrosulphide or caffeic acid phenethyl ester increased the activity of BKCa channels, but AUY did not. We further evaluated whether conductance of Ca(2+)-activated K(+) channels (IK(Ca)) influenced secretion of HCO3(-) and fluid in PDECs by using a modified Whitcomb-Ermentrout model. Simulation studies showed that an increase in IK(Ca) resulted in additional secretion of HCO3(-) and fluid by mimicking the effect of AUY in PDECs. Collectively, AUY can interact with the BKCa channel to largely increase IK(Ca) in PDECs. PMID:25953267

  14. Heat shock proteins in the retina: Focus on HSP70 and alpha crystallins in ganglion cell survival.

    PubMed

    Piri, Natik; Kwong, Jacky M K; Gu, Lei; Caprioli, Joseph

    2016-05-01

    Heat shock proteins (HSPs) belong to a superfamily of stress proteins that are critical constituents of a complex defense mechanism that enhances cell survival under adverse environmental conditions. Cell protective roles of HSPs are related to their chaperone functions, antiapoptotic and antinecrotic effects. HSPs' anti-apoptotic and cytoprotective characteristics, their ability to protect cells from a variety of stressful stimuli, and the possibility of their pharmacological induction in cells under pathological stress make these proteins an attractive therapeutic target for various neurodegenerative diseases; these include Alzheimer's, Parkinson's, Huntington's, prion disease, and others. This review discusses the possible roles of HSPs, particularly HSP70 and small HSPs (alpha A and alpha B crystallins) in enhancing the survival of retinal ganglion cells (RGCs) in optic neuropathies such as glaucoma, which is characterized by progressive loss of vision caused by degeneration of RGCs and their axons in the optic nerve. Studies in animal models of RGC degeneration induced by ocular hypertension, optic nerve crush and axotomy show that upregulation of HSP70 expression by hyperthermia, zinc, geranyl-geranyl acetone, 17-AAG (a HSP90 inhibitor), or through transfection of retinal cells with AAV2-HSP70 effectively supports the survival of injured RGCs. RGCs survival was also stimulated by overexpression of alpha A and alpha B crystallins. These findings provide support for translating the HSP70- and alpha crystallin-based cell survival strategy into therapy to protect and rescue injured RGCs from degeneration associated with glaucomatous and other optic neuropathies. PMID:27017896

  15. The spectrum of beta-thalassemia mutations in southern Thailand.

    PubMed

    Nopparatana, C; Panich, V; Saechan, V; Sriroongrueng, V; Nopparatana, C; Rungjeadpha, J; Pornpatkul, M; Laosombat, V; Fukumaki, Y

    1995-01-01

    Beta-thalassemia mutations in 282 alleles of 253 unrelated individuals originating from various provinces in the south of Thailand were characterized by dot blot hybridization, specific PCR-amplification and direct DNA sequencing. It was possible to characterize the mutations in 274 (97.2%) of alleles studied. Twelve different point mutations and two different large deletions of the beta-globin gene were identified. Seven common mutations, namely 4 bp deletion at codons 41/42. IVS1 position 5 (G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 position 1 (G-T), position -28 (A-G) and 3.5 kb deletion, accounted for about 91.5%. The mutations at mRNA cap site + 1 (A-C) and IVS1 position 1 (G-A), previously undescribed in Thailand, were found in 1 and 2 individuals, respectively. A novel mutation of 105 bp deletion at the 5' end of beta-globin gene was detected in a family originating from this area. The knowledge from this study should be useful for planning of genetic counseling and prenatal diagnosis programs for patients with beta-thalassemia in the south of Thailand. PMID:8629112

  16. NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Sensitivity and Resistance to Antitumor Quinones

    PubMed Central

    Siegel, David; Yan, Chao; Ross, David

    2012-01-01

    The quinone pharmacophore is present in many drug classes but is particularly common among antitumor drugs. Many quinones serve essentially as pro-drugs and exert their activities after reduction. Reduction of quinones may generate semiquinones or hydroquinones with subsequent generation of reactive oxygen radicals and oxidative stress, quinones can be designed so they lose a leaving group when reduced to the hydroquinone generating a reactive electrophile or the hydroquinone form of the molecule may have greater pharmacological activity than the parent quinone against a particular target. Enzyme systems that reduce quinones therefore become critically important in the pharmacological activity of this class of drugs. There are a number of enzyme systems that can catalyze reduction of quinones including cytochrome P450 reductase, cytochrome b5 reductase, NAD(P)H:quinone oxidoreductase 1 (NQO1), NAD(P)H:quinone oxidoreductase 2 (NQO2), carbonyl reductases, and thioredoxin reductase. In this context, one of the most extensively studied reductases has been NAD(P)H:quinone oxidoreductase 1 (NQO1). In this review we will focus on the role of NQO1 in the bioactivation of clinically important quinones mitomycin C, β-lapachone and 17AAG as well as the influence of the NQO1*2 polymorphism on the sensitivity and resistance to these agents. PMID:22209713

  17. Requirement of HDAC6 for activation of Notch1 by TGF-β1.

    PubMed

    Deskin, Brian; Lasky, Joseph; Zhuang, Yan; Shan, Bin

    2016-01-01

    TGF-β1 is enriched in the tumor microenvironment and acts as a key inducer of epithelial to mesenchymal transition (EMT) in lung cancer. The NOTCH signaling pathway is conserved across species and is an essential pathway for development, cell differentiation, and cancer biology. Dysregulation of Notch signaling is a common feature of non-small cell lung cancer (NSCLC) and is correlated with poor prognosis. Crosstalk exists between the NOTCH and TGF-β signaling pathways in EMT. Herein we report that histone deacetylase 6 (HDAC6) modulates TGF-β1-mediated activation of the Notch pathway. HDAC6, a primarily cytoplasmic deacetylase, mediates TGF-β1-induced EMT in human lung cancer cells. Inhibition of HDAC6 with a small molecule inhibitor, namely tubacin or with siRNA attenuated TGF-β1-induced Notch-1 signaling. We show that TGFβ-1-induced EMT is accompanied by rapid HDAC6-dependent deacetylation of heat shock protein 90 (HSP90). Consistently, inhibition of HSP90 with its small molecule inhibitor 17AAG attenuated expression of TGF-β1-induced Notch-1 target genes, HEY-1 and HES-1. These findings reveal a novel function of HDAC6 in EMT via mediating the TGF-β-Notch signaling cascade, and support HDAC6 as a key regulator of TGFβ-induced EMT in NSCLC. This work suggests that HDAC6 may be an attractive therapeutic target against tumor progression and metastasis. PMID:27499032

  18. Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain.

    PubMed

    Raman, Swetha; Suguna, Kaza

    2015-06-01

    Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source. PMID:26057797

  19. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  20. Hsp90 Inhibitors and Drug Resistance in Cancer: The Potential Benefits of Combination Therapies of Hsp90 Inhibitors and Other Anti-Cancer Drugs

    PubMed Central

    Lu, Xiangyi; Xiao, Li; Wang, Luan; Ruden, Douglas M.

    2012-01-01

    Hsp90 is a chaperone protein that interacts with client proteins that are known to be in the cell cycle, signaling and chromatin-remodeling pathways. Hsp90 inhibitors act additively or synergistically with many other drugs in the treatment of both solid tumors and leukemias in murine tumor models and humans. Hsp90 inhibitors potentiate the actions of anti-cancer drugs that target Hsp90 client proteins, including trastuzumab (Herceptin™) which targets Her2/Erb2B, as Hsp90 inhibition elicits the drug effects in cancer cell lines that are otherwise resistant to the drug. A Phase II study of the Hsp90 inhibitor 17-AAG and trastuzumab showed that this combination therapy has anticancer activity in patients with HER2-positive metastatic breast cancer progressing on trastuzumab. In this review, we discuss the results of Hsp90 inhibitors in combination with trastuzumab and other cancer drugs. We also discuss recent results from yeast focused on the genetics of drug resistance when Hsp90 is inhibited and the implications that this might have in understanding the effects of genetic variation in treating cancer in humans. PMID:22120678

  1. The neuroblastoma associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK translocated cancers

    PubMed Central

    Sasaki, Takaaki; Okuda, Katsuhiro; Zheng, Wei; Butrynski, James; Capelletti, Marzia; Wang, Liping; Gray, Nathanael S.; Wilner, Keith; Christensen, James G.; Demetri, George; Shapiro, Geoffrey I.; Rodig, Scott J.; Eck, Michael J.; Jänne, Pasi A.

    2011-01-01

    The ALK kinase inhibitor crizotinib (PF-02341066) is clinically effective in patients with ALK-translocated cancers, but its efficacy will ultimately be limited by acquired drug resistance. Here we report the identification of a secondary mutation in ALK, F1174L, as one cause of crizotinib resistance in a patient with an inflammatory myofibroblastic tumor (IMT) harbouring a RANBP2-ALK translocation who progressed while crizotinib therapy. When present in cis with an ALK translocation, this mutation (also detected in neuroblastomas) causes an increase in ALK phosphorylation, cell growth and downstream signaling. Furthermore, the F1174L mutation inhibits crizotinib mediated downregulation of ALK signaling and blocks apoptosis in RANBP2-ALK Ba/F3 cells. A chemically distinct ALK inhibitor, TAE684, or the HSP90 inhibitor 17-AAG are both effective in models harbouring the F1174L ALK mutation. Our findings highlight the importance of studying drug resistance mechanisms in order to develop effective clinical treatments for patients with ALK-translocated cancers. PMID:21030459

  2. Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression*

    PubMed Central

    Lin, Jeffrey J.; Hemenway, Charles S.

    2010-01-01

    AF9/MLLT3 contributes to the regulation of the gene encoding the epithelial sodium channel α, ENaCα, in renal tubular cells. Specifically, increases in AF9 protein lead to a reduction in ENaCα expression and changes in AF9 activity appear to be an important component of aldosterone signaling in the kidney. Whereas AF9 is found in the nucleus where it interacts with the histone H3 lysine 79 methyltransferase, Dot1, AF9 is also present in the cytoplasm. Data presented in this report indicate that the heat shock protein Hsp90 directly and specifically interacts with AF9 as part of an Hsp90-Hsp70-p60/Hop chaperone complex. Experimental manipulation of Hsp90 function by the inhibitor novobiocin, but not 17-AAG, results in redistribution of AF9 from a primarily nuclear to cytoplasmic location. Knockdown of Hsp90 with siRNA mimics the effect elicited by novobiocin. As expected, a shift in AF9 from the nucleus to the cytoplasm in response to Hsp90 interference leads to increased ENaCα expression. This is accompanied by a decrease in AF9 occupancy at the ENaCα promoter. Our data suggest that the interaction of Hsp90, Hsp70, and p60/Hop with AF9 is necessary for the proper subnuclear localization and activity of AF9. AF9 is among a growing number of nuclear proteins recognized to rely on the Hsp90 complex for nuclear targeting. PMID:20159978

  3. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. PMID:26722004

  4. Requirement of HDAC6 for activation of Notch1 by TGF-β1

    PubMed Central

    Deskin, Brian; Lasky, Joseph; Zhuang, Yan; Shan, Bin

    2016-01-01

    TGF-β1 is enriched in the tumor microenvironment and acts as a key inducer of epithelial to mesenchymal transition (EMT) in lung cancer. The NOTCH signaling pathway is conserved across species and is an essential pathway for development, cell differentiation, and cancer biology. Dysregulation of Notch signaling is a common feature of non-small cell lung cancer (NSCLC) and is correlated with poor prognosis. Crosstalk exists between the NOTCH and TGF-β signaling pathways in EMT. Herein we report that histone deacetylase 6 (HDAC6) modulates TGF-β1-mediated activation of the Notch pathway. HDAC6, a primarily cytoplasmic deacetylase, mediates TGF-β1-induced EMT in human lung cancer cells. Inhibition of HDAC6 with a small molecule inhibitor, namely tubacin or with siRNA attenuated TGF-β1-induced Notch-1 signaling. We show that TGFβ-1-induced EMT is accompanied by rapid HDAC6-dependent deacetylation of heat shock protein 90 (HSP90). Consistently, inhibition of HSP90 with its small molecule inhibitor 17AAG attenuated expression of TGF-β1-induced Notch-1 target genes, HEY-1 and HES-1. These findings reveal a novel function of HDAC6 in EMT via mediating the TGF-β-Notch signaling cascade, and support HDAC6 as a key regulator of TGFβ-induced EMT in NSCLC. This work suggests that HDAC6 may be an attractive therapeutic target against tumor progression and metastasis. PMID:27499032

  5. Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    PubMed Central

    Zhao, X M; Chen, Z; Zhao, J B; Zhang, P P; Pu, Y F; Jiang, S H; Hou, J J; Cui, Y M; Jia, X L; Zhang, S Q

    2016-01-01

    The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a key component of tumor necrosis factor (TNF)-induced necroptosis and plays a crucial role in necroptosis execution. However, the mechanisms that control MLKL activity are not completely understood. Here, we identify the molecular chaperone Hsp90 as a novel MLKL-interacting protein. We show that Hsp90 associates with MLKL and is required for MLKL stability. Moreover, we find that Hsp90 also regulates the stability of the upstream RIP3 kinase. Interference with Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3, resulting in their degradation by the proteasome pathway. Furthermore, we find that Hsp90 is required for TNF-stimulated necrosome assembly. Disruption of Hsp90 function prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis. Consistent with a positive role of Hsp90 in necroptosis, coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis. Our findings demonstrate that an efficient necrotic response requires a functional Hsp90. PMID:26866270

  6. High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

    PubMed Central

    Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

    2014-01-01

    Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active

  7. Comparison and validation of genomic predictors for anticancer drug sensitivity

    PubMed Central

    Papillon-Cavanagh, Simon; De Jay, Nicolas; Hachem, Nehme; Olsen, Catharina; Bontempi, Gianluca; Aerts, Hugo J W L; Quackenbush, John; Haibe-Kains, Benjamin

    2013-01-01

    Background An enduring challenge in personalized medicine lies in selecting the right drug for each individual patient. While testing of drugs on patients in large trials is the only way to assess their clinical efficacy and toxicity, we dramatically lack resources to test the hundreds of drugs currently under development. Therefore the use of preclinical model systems has been intensively investigated as this approach enables response to hundreds of drugs to be tested in multiple cell lines in parallel. Methods Two large-scale pharmacogenomic studies recently screened multiple anticancer drugs on over 1000 cell lines. We propose to combine these datasets to build and robustly validate genomic predictors of drug response. We compared five different approaches for building predictors of increasing complexity. We assessed their performance in cross-validation and in two large validation sets, one containing the same cell lines present in the training set and another dataset composed of cell lines that have never been used during the training phase. Results Sixteen drugs were found in common between the datasets. We were able to validate multivariate predictors for three out of the 16 tested drugs, namely irinotecan, PD-0325901, and PLX4720. Moreover, we observed that response to 17-AAG, an inhibitor of Hsp90, could be efficiently predicted by the expression level of a single gene, NQO1. Conclusion These results suggest that genomic predictors could be robustly validated for specific drugs. If successfully validated in patients’ tumor cells, and subsequently in clinical trials, they could act as companion tests for the corresponding drugs and play an important role in personalized medicine. PMID:23355484

  8. The antiproliferative activity of the heat shock protein 90 inhibitor IPI-504 is not dependent on NAD(P)H:quinone oxidoreductase 1 activity in vivo.

    PubMed

    Douglas, Mark; Lim, Alice R; Porter, James R; West, Kip; Pink, Melissa M; Ge, Jie; Wylie, Andrew A; Tibbits, Thomas T; Biggs, Kurtis; Curtis, Michael; Palombella, Vito J; Adams, Julian; Fritz, Christian C; Normant, Emmanuel

    2009-12-01

    IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic. PMID:19952119

  9. Heat shock protein 90 is involved in IL-17-mediated skin inflammation following thermal stimulation.

    PubMed

    Kim, Bo-Kyung; Park, Minhwa; Kim, Ji-Yon; Lee, Kyung-Ho; Woo, So-Youn

    2016-08-01

    The pathogenesis of inflammatory skin diseases involves interactions between immune cells and keratinocytes, including the T helper 17 (Th17)-mediated immune response. Several chemokines [chemokine (C-X-C motif) ligand (CXCL)1, CXCL5 and CXCL8] and antimicrobial peptides [β-defensin 1 (BD1), LL-37, S100A8 and S100A9] were transcriptionally upregulated in the keratinocyte cell line HaCaT upon stimulation with interleukin (IL)-17. Balneotherapy, the treatment of disease by bathing, is an alternative therapy that has frequently been used for the treatment of inflammatory skin diseases. Immersion in pools of thermal mineral water is often considered to have chemical, thermal, mechanical and immunomodulatory benefits. We examined the effect of thermal treatment on IL-17-mediated inflammation in a model of skin disease. As Act1 is required for IL-17 signaling and is a client protein of heat shock protein 90 (HSP90), we evaluated the effect of HSP90 inhibition on IL-17-mediated cytokine and antimicrobial peptide expression in keratinocytes following heat treatment. We found that after thermal stimulation, Act1 binding to HSP90α was significantly increased in the presence of IL-17 (100 ng/ml) and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, 1 µM). Antimicrobial peptide and chemokine expression generally increased after heat treatment; Act1 knockdown and 17‑AAG reversed this effect. These observations demonstrate the possible immunomodulatory effect of heat on keratinocytes during the progression of IL-17-mediated inflammatory skin diseases. PMID:27279135

  10. Both the Charged Linker Region and ATPase Domain of Hsp90 Are Essential for Rad51-Dependent DNA Repair

    PubMed Central

    Suhane, Tanvi; Laskar, Shyamasree; Advani, Siddheshwari; Roy, Nabamita; Varunan, Shalu; Bhattacharyya, Dibyendu

    2014-01-01

    The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time. PMID:25380755

  11. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    SciTech Connect

    Vuong, Tram Thu; Berger, Christian; Bertelsen, Vibeke; Rødland, Marianne Skeie; Stang, Espen; Madshus, Inger Helene

    2013-02-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub{sub 4}) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub{sub 4} chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub{sub 4} was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub{sub 4} was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub{sub 4} was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  12. A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells*

    PubMed Central

    Bailey, Tameka A.; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L.; Natarajan, Amarnath; Raja, Srikumar M.; Band, Vimla; Band, Hamid

    2014-01-01

    ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca2+-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling. PMID:25225290

  13. Current Understanding of HSP90 as a Novel Therapeutic Target: An Emerging Approach for the Treatment of Cancer.

    PubMed

    Haque, Absarul; Alam, Qamre; Alam, Mohammad Zubair; Azhar, Esam I; Sait, Khalid Hussain Wali; Anfinan, Nisrin; Mushtaq, Gohar; Kamal, Mohammad Amjad; Rasool, Mahmood

    2016-01-01

    Heat Shock Protein 90 (HSP90) is a ubiquitous molecular chaperone that is considered to be the most abundantly expressed protein in various human cancers such as breast, lung, colon, prostate, leukemia and skin. The master regulator, HSP90 plays a pivotal role in the conformational stabilization, maturation and activity of its various labile oncogenic client proteins such as p53, ErbB2, Bcr-Abl, Akt, Her-2, Cdk4, Cdk6, Raf-1 and v-Src in altered cells. Hence, making a guaranteed attempt to inhibit such a master regulator for cancer therapy appears to be a potential approach for combinatorial inhibition of numerous oncogenic signaling pathways simultaneously. Considerable efforts are being under way to develop novel molecular targets and its inhibitors that may block key signaling pathways involved in the process of tumorigenesis and metastasis. In this regards, HSP90 has acquired immense interest as a potent anticancer drug-target due to its key functional link with multiple signaling pathways involved in the process of cell proliferation and cell survival. Notably, geldanamycin and its derivatives (17-AAG, 17-DMAG) have shown quite encouraging results in inhibiting HSP90 function in several cancers and currently almost 17 drug candidates known to be target HSP90 are being under clinical trials either as single agents or combinatorial therapy. Hence, this review is an attempt to get new insight into novel drug target therapy by focusing on recent advances made in understanding HSP90 chaperone structure-function relationships, identification of new HSP90 client proteins and, more importantly, on the advancements of HSP90 targeted therapy based on various existing and emerging classical inhibitors. PMID:27013225

  14. Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells

    PubMed Central

    2012-01-01

    Background Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo. Results In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition. Conclusion These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer. PMID:22913813

  15. Antimyeloma activity of heat shock protein-90 inhibition.

    PubMed

    Mitsiades, Constantine S; Mitsiades, Nicholas S; McMullan, Ciaran J; Poulaki, Vassiliki; Kung, Andrew L; Davies, Faith E; Morgan, Gareth; Akiyama, Masaharu; Shringarpure, Reshma; Munshi, Nikhil C; Richardson, Paul G; Hideshima, Teru; Chauhan, Dharminder; Gu, Xuesong; Bailey, Charles; Joseph, Marie; Libermann, Towia A; Rosen, Neal S; Anderson, Kenneth C

    2006-02-01

    We show that multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy, is responsive to hsp90 inhibitors in vitro and in a clinically relevant orthotopic in vivo model, even though this disease does not depend on HER2/neu, bcr/abl, androgen or estrogen receptors, or other hsp90 chaperoning clients which are hallmarks of tumor types traditionally viewed as attractive clinical settings for use of hsp90 inhibitors, such as the geldanamycin analog 17-AAG. This class of agents simultaneously suppresses in MM cells the expression and/or function of multiple levels of insulin-like growth factor receptor (IGF-1R) and interleukin-6 receptor (IL-6R) signaling (eg, IKK/NF-kappaB, PI-3K/Akt, and Raf/MAPK) and downstream effectors (eg, proteasome, telomerase, and HIF-1alpha activities). These pleiotropic proapoptotic effects allow hsp90 inhibitors to abrogate bone marrow stromal cell-derived protection on MM tumor cells, and sensitize them to other anticancer agents, including cytotoxic chemotherapy and the proteasome inhibitor bortezomib. These results indicate that hsp90 can be targeted therapeutically in neoplasias that may not express or depend on molecules previously considered to be the main hsp90 client proteins. This suggests a more general role for hsp90 in chaperoning tumor- or tissue-type-specific constellations of client proteins with critical involvement in proliferative and antiapoptotic cellular responses, and paves the way for more extensive future therapeutic applications of hsp90 inhibition in diverse neoplasias, including MM. PMID:16234364

  16. Role of ARF6, Rab11 and external Hsp90 in the trafficking and recycling of recombinant-soluble Neisseria meningitidis adhesin A (rNadA) in human epithelial cells.

    PubMed

    Bozza, Giuseppe; Capitani, Mirco; Montanari, Paolo; Benucci, Barbara; Biancucci, Marco; Nardi-Dei, Vincenzo; Caproni, Elena; Barrile, Riccardo; Picciani, Benedetta; Savino, Silvana; Aricò, Beatrice; Rappuoli, Rino; Pizza, Mariagrazia; Luini, Alberto; Sallese, Michele; Merola, Marcello

    2014-01-01

    Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells. PMID:25347845

  17. Gateways to clinical trials.

    PubMed

    Tomillero, A; Moral, M A

    2010-09-01

    Aclidinium bromide, AE-37, Alemtuzumab, AMA1-C1/ISA 720, Amlodipine besylate/atorvastatin calcium, Arachidonic acid, Arbaclofen placarbil, Aripiprazole, ARQ-621, Azelnidipine, Azilsartan medoxomil potassium; Bevacizumab, Biphasic insulin aspart, Bortezomib; Choriogonadotropin alfa, CTS-1027; Dapagliflozin, Dasatinib, Deforolimus, Degarelix acetate, Denufosol tetrasodium, Desvenlafaxine succinate, Dronedarone hydrochloride, Duloxetine hydrochloride, Dutasteride; Enfuvirtide, Entecavir, Etaracizumab, Everolimus, Exenatide, Ezetimibe; Ferric carboxymaltose, Fludarabine, Foretinib; Gefitinib, GFT-505, GSK-256066; HPV-6/11/16/18, HuM195/rGel, HyperAcute-Lung cancer vaccine; I5NP, Imatinib mesylate, Imexon, Insulin detemir, Insulin glargine, Ivabradine hydrochloride; L2G7, Lacosamide, Lapatinib ditosylate, Lenalidomide, Lidocaine/prilocaine, Liposomal vincristine, Liraglutide, Lixivaptan; Meningococcal (groups A, C, Y and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine, Methoxy polyethylene glycol-epoetin-β, Mirabegron, Morphine/oxycodone, MR Vaccine, MSC-1936369B, Mycophenolic acid sodium salt; Narlaprevir, N-Desmethylclozapine; Ocriplasmin, Olaparib, Olmesartan medoxomil, Olmesartan medoxomil/azelnidipine, ONO-5334, ONO-8539; Palifermin, Panitumumab, Pardoprunox hydrochloride, PCV7, Peginterferon alfa-2a, Peginterferon alfa-2b, Pemetrexed disodium, Pexelizumab, PF-337210, Pitavastatin calcium; Raltegravir potassium, Recombinant interleukin-7, Regadenoson, Reniale, Roflumilast, Rosuvastatin calcium; Safinamide mesilate, SB-1518, SCH-527123, Selumetinib, Sipuleucel-T, Solifenacin succinate, Sorafenib, Sunitinib malate; Tadalafil, Talaporfin sodium, Tanespimycin, Technosphere/Insulin, Telaprevir, Telatinib, Telcagepant, Telmisartan/hydrochlorothiazide, Teriparatide, Testosterone transdermal gel, TH-302, Tiotropium bromide, Tocilizumab, Trabedersen, Tremelimumab; Valsartan/amlodipine besylate, Vernakalant hydrochloride, Visilizumab, Voreloxin, Vorinostat. PMID

  18. Gateways to clinical trials.

    PubMed

    Tomillero, A; Moral, M A

    2009-11-01

    Abacavir sulfate/lamivudine, Adalimumab, AdCD40L, Adefovir, Adefovir dipivoxil, Ambrisentan, Amlodipine, Amlodipine besylate/olmesartan medoxomil, AN-2728, Apixaban, Aripiprazole, Armodafinil, Atazanavir sulfate, Atomoxetine hydrochloride, Atrasentan, Azacitidine, Bevacizumab, Blinatumomab, Bortezomib, Bosentan, Carfilzomib, Caspofungin acetate, Cediranib, Cetuximab, Choriogonadotropin alfa, Clevudine, Clindamycin phosphate/benzoyl peroxide, Clofarabine, Daidzeol, Darunavir, Dasatinib, Decitabine, Deferasirox, Deforolimus, Degarelix acetate, Denenicokin, Dexlansoprazole, Duloxetine hydrochloride, Elacytarabine, Enfuvirtide, Enoxaparin, Entecavir, Eribulin mesilate, Erlotinib hydrochloride, Escitalopram oxalate, Eslicarbazepine acetate, Eszopiclone, Etravirine, Ezetimibe/simvastatin, Forodesine hydrochloride, Fosamprenavir calcium, Gefitinib, Gemtuzumab ozogamicin, Golimumab, Imatinib mesylate, Imetelstat, Insulin gl'argine, Insulin glulisine, Interferon alfa-2b XL, Ivabradine hydrochloride, Lacosamide, Lenalidomide, Lintuzumab, Liposomal adriamycin, Liposomal belotecan, Liposome-encapsulated fentanyl, Lopinavir/ritonavir, Lutropin alfa, LY-207320, Maraviroc, Mecasermin, MKC-253, MP-470, NGR-TNF, Nilotinib hydrochloride monohydrate, Ofatumumab, Olmesartan medoxomil, Omacetaxine mepesuccinate, PAN-811, Panobinostat, Pegfilgrastim, Peginterferon alfa-2a, Peginterferon alfa-2b, Peginterferon alfa-2b/ribavirin, Pemetrexed disodium, Perospirone hydrochloride, PF-734200, Phentermine/topiramate, Pimecrolimus, Pitavastatin calcium, Plerixafor hydrochloride, Pregabalin, Raltegravir potassium, Ramelteon, Ranibizumab, Recombinant Bet V1, Recombinant human insulin, Regadenoson, rhITF, Romidepsin, Rosuvastatin calcium, Ruboxistaurin hydrochloride, Rufinamide, Sapropterin dihydrochloride Saracatinib, SB-73, SC-599, Seliciclib, Sirolimus-eluting stent, Sorafenib, Sunitinib malate, Tadalafil, Tanespimycin, Tapentadol hydrochloride, Tegaserod maleate, Telbivudine, Tenofovir

  19. Gateways to clinical trials.

    PubMed

    Tomillero, A; Moral, M A

    2008-10-01

    Gateways to clinical trials is a guide to the most recent trials in current literature and congresses. The data in the following tables has been retrieved from the Clinical Trials Knowledge Area of Prous Science Integrity(R), the drug discovery and development portal, http://integrity.prous.com. This issue focuses on the following selection of drugs: (+)-Dapoxetine hydrochloride, (S)-Tenatoprazole sodium salt monohydrate 19-28z, Acotiamide hydrochloride hydrate, ADV-TK, AE-37, Aflibercept, Albinterferon alfa-2b, Aliskiren fumarate, Asenapine maleate, Axitinib; Bavituximab, Becatecarin, beta-1,3/1,6-Glucan, Bevacizumab, Bremelanotide; Calcipotriol/betamethasone dipropionate, Casopitant mesylate, Catumaxomab, CDX-110, Cediranib, CMD-193, Cositecan; Darinaparsin, Denosumab, DP-b99, Duloxetine hydrochloride; E75, Ecogramostim, Elacytarabine, EMD-273063, EndoTAG-1, Enzastaurin hydrochloride, Eplerenone, Eribulin mesilate, Esomeprazole magnesium, Etravirine, Everolimus, Ezetimibe; Faropenem daloxate, Febuxostat, Fenretinide; Ghrelin (human); I-131 ch-TNT-1/B, I-131-3F8, Iclaprim, Iguratimod, Iloperidone, Imatinib mesylate, Inalimarev/Falimarev, Indacaterol, Ipilimumab, Iratumumab, Ispinesib mesylate, Ixabepilone; Lapatinib ditosylate, Laquinimod sodium, Larotaxel dehydrate, Linezolid, LOR-2040; Mapatumumab, MKC-1, Motesanib diphosphate, Mycophenolic acid sodium salt; NK-012; Olanzapine pamoate, Oncolytic HSV, Ortataxel; Paclitaxel nanoparticles, Paclitaxel poliglumex, Paliperidone palmitate, Panitumumab, Patupilone, PCV-9, Pegfilgrastim, Peginterferon alfa-2a, Peginterferon alfa-2b, Pertuzumab, Picoplatin, Pimavanserin tartrate, Pimecrolimus, Plerixafor hydrochloride, PM-02734, Poly I:CLC, PR1, Prasugrel, Pregabalin, Progesterone caproate, Prucalopride, Pumosetrag hydrochloride; RAV-12, RB-006, RB-007, Recombinant human erythropoietin alfa, Rimonabant, Romidepsin; SAR-109659, Satraplatin, Sodium butyrate; Tadalafil, Talampanel, Tanespimycin, Tarenflurbil, Tariquidar

  20. Gateways to clinical trials.

    PubMed

    Tomillero, A; Moral, M A

    2009-03-01

    ABT-869, Acadesine, Acetylsalicylic acid/omeprazole, Adefovir, Adefovir dipivoxil, AEG-35156, Agatolimod sodium, Albiglutide, Alemtuzumab, Alipogene tiparvovec, Alogliptin benzoate, AMG-386, Amrubicin hydrochloride, Apremilast, Aripiprazole, Asoprisnil, Atorvastatin/fenofibrate, AVN-944, Axitinib; Belinostat, Bevacizumab, BHT-3021, BI-2536, Biapenem, Bilastine, Biphasic insulin aspart, Blinatumomab, Bortezomib, Bosentan; Catumaxomab, CD-NP, Cediranib, Certolizumab pegol, Cetuximab, Choline fenofibrate, Ciclesonide, CK-1827452,Clevudine, Clofarabine, CSL-360, CYT-997; Dapagliflozin, Darinaparsin, Denosumab, Densiron 68, Desloratadine, Dulanermin; Edoxaban tosilate, Emtricitabine, Entecavir, Erlotinib hydrochloride, Everolimus, Exenatide, Ezetimibe, Ezetimibe/simvastatin; Fidaxomicintiacumiv, Fulvestrant; G-207, GCR-8015, Gefitinib, Ghrelin (human), Glufosfamide; HPV16L1E7CVLP; Ibutamoren mesilate, Imatinib mesylate, Insulin detemir, Insulin glargine, Iodine (I131) tositumomab, Istaroxime, ITMN-191, Ixabepilone; JZP-4, Lenalidomide; Levetiracetam, Linaclotide acetate, Liposomal cytarabine/daunorubicin, Liposomal doxorubicin, Liraglutide, LY-518674; Milatuzumab, MMR-V, Motesanib diphosphate, Mycophenolic acid sodium salt; Niacin/simvastatin; Obatoclax mesylate, Odanacatib; Paclitaxel nanoparticles, Paclitaxel-eluting stent, Pazufloxacin, PBT-2, Pegfilgrastim, Peginterferon alfa-2a, Peginterferon alfa-2b, Peginterferon alfa-2b/ribavirin, Pemetrexed disodium, Perampanel, PfCP2.9, Pitavastatin calcium, Poly I:CLC, Pomalidomide, Pralatrexate, Pramlintide acetate, Prucalopride; rhGAD65, Roflumilast; RTS,S/AS02D; SCH-530348, Semagacestat, Sirolimus-eluting coronary stent, Sirolimus-Eluting Stent, SIR-Spheres, Sivelestat sodium hydrate, Sorafenib, Sunitinib malate; Tadalafil, Tafluprost, Tanespimycin, Teduglutide, Telaprevir, Telbivudine, Tenofovir disoproxil fumarate, Tiotropium bromide, TMC-435350, Tositumomab/iodine (I131) tositumomab, Travoprost/timolol, Triciribine

  1. Gateways to clinical trials.

    PubMed

    Tomillero, A; Moral, M A

    2009-09-01

    mesilate, Recombinant human relaxin H2, rhGAD65, Rivaroxaban, Rosuvastatin calcium, Rotigotine; Saxagliptin, SCH-530348, Sirolimus-eluting stent, SLIT-amikacin, Sorafenib, Sotrastaurin, SR-16234, Sulforaphane; Tadalafil, Tanespimycin, Tapentadol hydrochloride, Teriparatide, Tesofensine, Tiotropium bromide, Tipifarnib, Tirapazamine, TMC-207, Tocilizumab, Tolvaptan, Tosedostat, Treprostinil sodium; Ustekinumab; Varespladib methyl, Vicriviroc, Vildagliptin, Vildagliptin/metformin hydrochloride, Volociximab, Voriconazole; Ziconotide, Ziprasidone hydrochloride. PMID:19907722

  2. Gateways to clinical trials.

    PubMed

    Bayes, M; Rabasseda, X; Prous, J R

    2007-10-01

    , Solifenacin succinate, Sunitinib malate; Tadalafil, Talnetant, Tanespimycin, Taxus, Tegaserod maleate, Telmisartan/hydrochlorothiazide, Tenofovir disoproxil fumarate/emtricitabine, Teriparatide, tgAAC-94, Tiotropium bromide, Tocilizumab, Tolvaptan, Trimethoprim; Vardenafil hydrochloride hydrate, Vatalanib succinate, Vinflunine, Voriconazole, VX-680; XL-880; Yttrium 90 (90Y) ibritumomab tiuxetan. PMID:18040531

  3. Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus

    PubMed Central

    Shinde, Sunita N.; Hua, Yun; Shun, Tong Ying; Lazo, John S.; Day, Billy W.

    2012-01-01

    Abstract Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to

  4. Geldanamycin and Its Derivatives Inhibit the Growth of Myeloma Cells and Reduce the Expression of the MET Receptor.

    PubMed

    Jurczyszyn, Artur; Zebzda, Anna; Czepiel, Jacek; Perucki, William; Bazan-Socha, Stanisława; Cibor, Dorota; Owczarek, Danuta; Majka, Marcin

    2014-01-01

    down-regulation of the MET receptor. The GA derivatives tested, despite their modifications still retain strong anticancer properties. Specifically, two analogues of GA, 17AEP-GA and 17DMAG due to their properties can be more effective and safer chemotherapeutic agents than 17AAG, which is currently used and described in literature. PMID:24959301